Control of protein glycosylation by culture medium supplementation and cell culture process parameters

ABSTRACT

The present invention pertains to a cell culture medium comprising media supplements that are shown to control recombinant protein glycosylation and/or cell culture in a controlled or modulated (shifted) temperature to control recombinant protein glycosylation and/or cell culture with controlled or modulated seed density to control recombinant protein glycosylation, and methods of using thereof. The present invention further pertains to a method of controlling or manipulating glycosylation of a recombinant protein of interest in a large scale cell culture.

This application is a continuation application of U.S. application Ser.No. 15/000,522, filed Jan. 19, 2016 which is a continuation applicationof U.S. application Ser. No. 14/625,559, filed Feb. 18, 2015 and acontinuation-in-part application of International Application No.PCT/US2014/051727, filed Aug. 19, 2014, said International ApplicationNo. PCT/US2014/051727 claims the benefit of U.S. Provisional ApplicationNo. 61/867,592, filed Aug. 19, 2013, all of which are hereinincorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains to a cell culture medium comprising mediasupplements that are shown to control recombinant protein glycosylationand/or cell culture in a controlled or modulated (shifted) temperatureto control recombinant protein glycosylation, and/or cell culture with acontrolled or modulated seed density, and methods of using thereof. Thepresent invention further pertains to a method of controlling ormanipulating glycosylation of a recombinant protein of interest in alarge scale cell culture, comprising supplementing the cell culture withadditives, such as mycophenolic acid, mycophenolic acid acylglucuronide, insulin, copper II sulfate, glucosamine, galactose,guanine, hypoxanthine, thymidine, or mixtures thereof, and/orcontrolling or modulating (shifting) the cell culture temperature,and/or controlling or modulating the cell culture seed density, or acombination thereof.

Background Art

Over the last few decades, much research has focused on the productionof therapeutic recombinant proteins, e.g., monoclonal antibodies, andthe work has taken a variety of angles. While much work in theliterature has utilized media containing sera or hydrolysates,chemically defined media were also developed in order to eliminate theproblematic lot-to-lot variation of complex components (Luo and Chen,Biotechnology and Bioengineering 97(6):1654-1659 (2007)). An improvedunderstanding of the cell culture has permitted a shift to chemicallydefined medium without compromising on growth, viability, titer, etc. Todate optimized chemically defined processes have been reported withtiters as high as 7.5-10 g/L (Huang et al., Biotechnology Progress26(5):1400-1410 (2010); Ma et al., Biotechnology Progress25(5):1353-1363 (2009); Yu et al., Biotechnology and Bioengineering108(5):1078-1088 (2011)). In general, the high titer chemically definedprocesses are fed batch processes with cultivation times of 11-18 days.The process intensification has been achieved without compromisingproduct quality while maintaining relatively high viabilities.

Achievement of a robust, scalable production process includes more thanincreasing the product titer while maintaining high product quality. Theprocess must also predictably require the main carbohydrate sourceremain constant, such that the feeding strategy does not need to changeacross scales. As many processes use glucose as the main carbohydrate,and have lactate and ammonium as the main byproducts, the time course ofthese three critical chemicals should also scale.

A number of reports have demonstrated mammalian host cell-specificprocessing of N-glycans associated with recombinant proteins (James etal., Bio/Technology, 13:592-596 (1995); Lifely et al., Glycobiology,5:813-822 (1995)). These differences may be important for therapeuticproteins as they can directly alter the antigenicity, rate of clearancein vivo, and stability of recombinant proteins (Jenkins et al., NatureBiotechnol. 14:975-981 (1996)). Thus, it is important not only to beable to characterize glycans bound to a therapeutic recombinant proteinto predict the consequences for in vivo safety and efficacy, but also tounderstand the cellular controls underpinning glycan processing in apotential host cell enabling the implementation of appropriatestrategies to control cellular glycosylation (Grabenhosrt et al.,Glycoconjug. J., 16:81-97 (1999); James and Baker, Encyclopedia ofbioprocess technology: Fermentation, biocatalysis and bioseparation. NewYork: John Wiley & Sons. p. 1336-1349 (1999)).

Thus, there is a need in the art for identification of methods that canpredictably control glycosylation of proteins of interest.

BRIEF SUMMARY OF THE INVENTION

The present invention pertains to a method of altering the glycosylationpattern of a recombinant glycoprotein produced in cell culturecomprising: culturing eukaryotic cells engineered to express arecombinant glycoprotein of interest in a cell culture medium, whereinthe cell culture medium is supplemented with an additive comprising oneor more of mycophenolic acid, mycophenolic acid acyl glucuronide,insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine,glucosamine, or galactose; wherein the glycosylation pattern of therecombinant glycoprotein of interest is altered relative to the samerecombinant glycoprotein produced by the same cells in the same cellculture medium without the additive.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: culturing eukaryotic cellsengineered to express a recombinant glycoprotein of interest in a cellculture medium, wherein the cell culture medium is supplemented with anadditive comprising one or more of mycophenolic acid, mycophenolic acidacyl glucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine,guanine, glucosamine, or galactose; wherein the glycosylation pattern ofthe recombinant glycoprotein of interest is altered to better resemblethe glycosylation pattern of a reference sample of the glycoprotein.

In a further embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: supplementing the culture medium ofa cell culture of eukaryotic cells engineered to express a recombinantglycoprotein of interest with an additive comprising one or more ofmycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper(II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, orgalactose; wherein the glycosylation pattern of the recombinantglycoprotein of interest is altered relative to the same recombinantglycoprotein produced by the same cells in the same cell culture mediumwithout the additive.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: supplementing the culture medium ofa cell culture of eukaryotic cells engineered to express a recombinantglycoprotein of interest with an additive comprising one or more ofmycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper(II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, orgalactose; wherein the glycosylation pattern of the recombinantglycoprotein of interest is altered to better resemble the glycosylationpattern of a reference sample of the glycoprotein.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating the cellculture temperature. In one embodiment, the method comprises increasingthe cell culture temperature. In another embodiment, the methodcomprising decreasing the cell culture temperature. In one embodimentthe method of altering the glycosylation pattern of a recombinantglycoprotein produced in cell culture comprising controlling ormodulating cell culture temperature together with supplementing theculture medium of a cell culture of eukaryotic cells engineered toexpress a recombinant glycoprotein of interest with an additivecomprising one or more of mycophenolic acid, mycophenolic acid acylglucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine,guanine, glucosamine, or galactose.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating the cellculture seed density. In one embodiment, the method comprises increasingthe cell culture seed density. In another embodiment, the methodcomprising decreasing the cell culture seed density. In one embodimentthe method of altering the glycosylation pattern of a recombinantglycoprotein produced in cell culture comprising controlling ormodulating cell culture seed density together with supplementing theculture medium of a cell culture of eukaryotic cells engineered toexpress a recombinant glycoprotein of interest with an additivecomprising one or more of mycophenolic acid, mycophenolic acid acylglucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine,guanine, glucosamine, or galactose.

In one embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating cellculture temperature together with supplementing the culture medium withmycophenolic acid.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating cellculture temperature together with supplementing the culture medium withmycophenolic acid acyl glucuronide.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating cellculture seed density together with supplementing the culture medium withmycophenolic acid.

In another embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: controlling or modulating cellculture seed density together with supplementing the culture medium withmycophenolic acid acyl glucuronide.

In another embodiment, the present invention pertains to furtherrecovering the recombinant glycoprotein of interest from the cellculture.

In another embodiment, the alteration of the glycosylation pattern ofthe recombinant glycoprotein of interest comprises one or more of areduced level of afucosylation, a reduced level of galactosylation, areduced level of galactose-alpha-1,3-galactose (α-gal), a reduced levelof N-glycolylneuraminic acid (NGNA), reduced FcγRIIIa binding, reducedantibody-dependent cell-mediated cytotoxicity, or an increased N-glycancharge. In another embodiment, the alteration of the glycosylationpattern of the recombinant glycoprotein of interest comprises reducedlevel of afucosylation.

In another embodiment, the alteration of the glycosylation pattern ofthe recombinant glycoprotein of interest comprises one or more ofincreased level of afucosylation, an increased level of galactosylation,an increased level of galactose-alpha-1,3-galactose (α-gal), anincreased level of N-glycolylneuraminic acid (NGNA), increased FcγRIIIabinding, increased antibody-dependent cell-mediated cytotoxicity, or anincreased N-glycan charge. In one embodiment, the alteration of theglycosylation pattern of the recombinant glycoprotein of interestcomprises increased level of afucosylation.

In one embodiment, the alteration of the glycosylation pattern of therecombinant glycoprotein of interest is achieved while minimizing one ormore undesirable side effects.

In a preferred embodiment, the present invention pertains to a method ofaltering the glycosylation pattern of a recombinant glycoproteinproduced in cell culture comprising: supplementing the culture mediumwith mycophenolic acid (MPA), or supplementing the culture medium withmycophenolic acid and insulin, or supplementing the culture medium withmycophenolic acid and galactose, supplementing the culture medium withmycopphenolic acid, insulin and galactose, or mycophenolic acid acylglucuronide (acMPAG), or supplementing the culture medium withmycophenolic acid and insulin, or supplementing the culture medium withmycophenolic acid acyl glucuronide and galactose, supplementing theculture medium with mycophenolic acid acyl glucuronide, insulin andgalactose, or supplementing the culture medium with copper (II) sulfate,or supplementing the culture medium with copper (II) sulfate, galactoseand hypoxanthine, or supplementing the culture medium with glucosamineand galactose, or modulated cell culture temperature and supplementingthe culture medium with mycophenolic acid, or modulated seed density andsupplementing the culture medium with mycophenolic acid, or modulatedcell culture temperature and supplementing the culture medium withmycophenolic acid acyl glucuronide, or modulated seed density andsupplementing the culture medium with mycophenolic acid acylglucuronide.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A and FIG. 1B. Effect of glucosamine and galactose on the growth(FIG. 1A) and viability (FIG. 1B) of fed-batch shake flask cultures.Glucosamine was delivered as two 10 mM boli, each on Day 2 and Day 4 inboth the glucosamine alone condition and the glucosamine/galactosecombination condition. In the combination condition, galactose wasdelivered as part of the 5×-concentrated DMEM/F12 feed media at aconcentration of 20 g/L in the feed. The Day 14 final concentration ofgalactose was 6 g/L, due to 30% feeding.

FIG. 2A and FIG. 2B. Effect of glucosamine and galactose on the growth(FIG. 2A) and viability (FIG. 2B) of fed-batch shake flask cultures ofimmunoadhesin-expressing CHO cells. Glucosamine was delivered as 10 mMboli on Day 6 for the 10 mM conditions and again on Day 8 for the 20 mMcondition. In the glucosamine and galactose combination conditions,galactose was delivered as part of the 5×-concentrated DMEM/F12 feedmedia at a concentration of 20 g/L in the feed. The Day 14 finalconcentration of galactose was 4 g/L, due to 20% feeding.

FIG. 3A and FIG. 3B. Effect of hypoxanthine and guanine on the growth(FIG. 3A) and viability (FIG. 3B) of fed-batch shake flask cultures ofimmunoadhesin-expressing CHO cells. Copper (II) sulfate was delivered aspart of the 5×-concentrated DMEM/F12 feed media at a concentration of 1mM in the feed media. The Day 14 final concentration of CuSO₄ in theculture was 0.2 mM due to 20% feeding. Hypoxanthine was delivered as a 1mM bolus from a 200 mM stock solution on Day 6 after the temperatureshift. Guanine was delivered as a 0.2 mM bolus from a 100 mM stocksolution on Day 6 after the temperature shift.

FIG. 4A and FIG. 4B. Effect of copper (II) sulfate on the growth (FIG.4A) and viability (FIG. 4B) of fed-batch shake flask cultures ofimmunoadhesin-expressing CHO cells. Copper (II) sulfate was delivered aspart of the 5×-concentrated DMEM/F12 feed media at a concentration of 1mM in the feed media. The Day 14 final concentration of CuSO₄ in theculture was 0.2 mM, due to 20% feeding. Alternatively, copper (II)sulfate was delivered as 0.2 mM and 0.5 mM boli on Day 6 from a stocksolution.

FIG. 5A and FIG. 5B. Effect of copper (II) sulfate, hypoxanthine,glucosamine, and galactose on the growth (FIG. 5A) and viability (FIG.5B) of 3 L fed-batch bioreactor cultures of immunoadhesin-expressing CHOcells. Copper (II) sulfate was delivered as part of the 5×-concentratedDMEM/F12 feed media at a concentration of 1 mM in the feed media. TheDay 14 final concentration of CuSO₄ in the culture was 0.25 mM, due to25% feeding. In the CuSO4, glucosamine, and galactose combinationconditions, copper (II) sulfate was delivered as part of the5×-concentrated DMEM/F12 feed media at a concentration of 1 mM in thefeed media. The Day 14 final concentration of CuSO₄ in the culture was0.22 mM, due to 22% feeding. Glucosamine was delivered as two 10 mMboli, each on Day 6 and Day 8. Galactose was delivered as part of the5×-concentrated DMEM/F12 feed media from Day 6-14 at a concentration of20 g/L in the feed. The Day 14 final concentration of galactose was 2.4g/L, due to 12% feeding from Day 6-14. In the CuSO₄ and hypoxanthinecondition, copper (II) sulfate and hypoxanthine were delivered as partof the 5×-concentrated DMEM/F12 feed media at a concentration of 1 mMand 3 mM, respectively, in the feed media. The Day 14 finalconcentrations of CuSO₄ and hypoxanthine in the culture were 0.23 mM and0.7 mM, due to 23% feeding.

FIG. 6A and FIG. 6B. Effect of copper (II) sulfate andHypoxanthine-Thymidine (HT) supplement on the growth (FIG. 6A) andviability (FIG. 6B) of immunoadhesin-expressing CHO cells in fed-batchshake flasks. The basal media was glutamine-free CHOM45+10 mg/L insulinand seeded at 9×10⁵ vc/mL. Copper (II) sulfate, hypoxanthine, andthymidine were delivered as part of the 5×-concentrated DMEM/F12 feedmedia at a concentration of 1 mM, 1 mM, and 0.16 mM respectively in thefeed media for the HT Low condition and 1 mM, 3 mM, and 0.5 mMrespectively in the feed media for the HT High condition. The Day 14final concentration of CuSO₄, hypoxanthine, and thymidine in the culturewas 0.30 mM, 0.30 mM, and 0.05 mM respectively for the HT Low conditionand 0.30 mM, 1 mM, and 0.16 mM respectively for the HT High conditiondue to 30% feeding.

FIG. 7A and FIG. 7B. Impact of Mycophenolic acid (MPA) on afucosylationin cell lines (FIG. 7A and FIG. 7B showing two different cell lines)derived from DUXB11. The cells were cultured in 3 L bioreactorrespectively for 7 days following the platform process. On day 7, thecells were divided into several 1 L shake flasks with 200 mL workingvolume and then dosed with various amounts of MPA (0 μM, 1 μM, 10 μM, 30μM). After dosing MPA, shake flask fed-batch is conducted until day-3harvest. Filtered supernatant samples were analyzed for titer andN-glycan analysis for afucosylation level.

FIG. 8. Impact of the timing of supplementation with Mycophenolic acid(MPA) on afucosylation of DUXB11 cell line in 3 L bioreactors (A, B, C,and D) with the same seed density following the platform process. Whenthe viable cell density (VCD) reached its peak (Day 5 in the study),fixed 5 μM of MPA was added in bioreactor A. At the same time, MPA wasadded in bioreactor B based on the VCD value to make sure MPA per cellis the same as the platform process to eliminate the possible impact ofdifferent cell growth. The next day (one day past peak VCD day, D6 inthis study), fixed 5 μM of MPA was added in bioreactor C and MPA wasadded in bioreactor D based on its D6 VCD value to make sure MPA percell is the same as the platform process to eliminate the possibleimpact of different cell growth. All bioreactors were harvested on D13.The filtered supernatant samples were analyzed for titer and N-glycananalysis for afucosylation level.

FIG. 9A and FIG. 9B. Impact of seed density growth (FIG. 9A) andviability (FIG. 9B) of DUXB11 cell line that was cultured in 3 Lbioreactors. The cells were grown with different seed densities (low,regular, high) following the platform process. Fixed 5 μM of MPA wasadded on Day 5 in all bioreactors. Different cell performances wereobserved due to different seed density.

FIG. 10. Impact of seed density on afucosylation in DUXB11 cell linethat was cultured in 3 L bioreactors. The cells were grown withdifferent seed densities (low, regular, high) following the platformprocess. Fixed 5 μM of MPA was added on Day 5 in all bioreactors.Different cell performances were observed due to different seed density.Filtered supernatant samples were analyzed for titer and N-glycananalysis for afucosylation level.

FIG. 11. Impact of insulin on afucosylation on DUXB11 cell line. Thecells was cultured in several 3 L bioreactors with the different insulinconcentration additions following the platform process. Fixed 5 μM ofMPA was added on Day 9 in all bioreactors and harvested on the same day.Filtered supernatant samples were analyzed for titer and N-glycananalysis for afucosylation level.

FIG. 12. Impact of timing of temperature shift on afucosylation.

FIG. 13. Impact of timing of temperature shift on afucosylation.

FIG. 14A and FIG. 14B. Factorial DOE study showing impact of timing oftemperature shift on afucosylation. FIG. 14A shows afucosylation levelafter temperature shift on various days. FIG. 14B shows afucosylationlevel after shifting to various temperatures on days 4 and 6.

FIG. 15A-FIG. 15C and FIG. 16A-FIG. 16C. Impact of Mycophenolic acid(MPA) on growth (FIG. 15A), viability (FIG. 15B), and titer (FIG. 15C)of ED003 cells in a shake flask. Impact of Mycophenolic acid (MPA) ongrowth (FIG. 16A), viability (FIG. 16B), and titer (FIG. 16C) of BIIB603cells in a shake flask.

FIG. 17A-FIG. 17C. Impact of Mycophenolic acid (MPA) on viability (FIG.17A), growth (FIG. 17B), and titer (FIG. 17C) in a bioreactor.

FIG. 18. Impact of Mycophenolic acid (MPA) and Mycophenolic acid acylglucuronide (acMPAG) on afucosylation in BIIB603 cell line. The cellswere scaled up to 5 L BR for fed-batch production process. On day 5,temperature was shifted from 37° C. to 31° C. On day 6, cell culture wasdrained from the bioreactor and divided into several 500 mL shake flaskswith 100 mL working volume and then dosed with various amounts of MPA (0μM, 20 μM, 40 μM) or acMPAG (20 μM) in duplicates. After dosing, theshake flasks were cultured another 4 days at 31° C., 5% CO₂ and 150 rpmwith fed-batch process and then harvested. The supernatant was sent forPQ assay (N-glycan analysis) for afucosylation level.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the recognition that cell culturemedia supplemented with mycophenolic acid, mycophenolic acid acylglucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine,guanine, glucosamine (GlcN), galactose, or mixtures thereof provide theability to control and manipulate the glycolsylation patterns ofrecombinant glycoproteins produced in eukaryotic cell cultures. Thepresent invention is also based on the recognition that a change in thecell culture temperature or cell culture seed density provides theability to control and manipulate the glycosylation patterns ofrecombinant glycoproteins produced in the eukaryotic cell cultures. Suchglyclosylation patterns include, without limitation, the level ofafucosylation, the level of galactosylation, the level of N-glycancharge, the level of N-glycolylneuraminic acid (NGNA), the level ofgalactose-alpha-1,3-galactose (α-gal), the level of antibody-dependentcell-mediated cytotoxicity (ADCC), and/or the level of FcγRIIIa binding.As the culture supplements have differential effects on bothglycosylation patterns and culture conditions, addition of the varioussupplements alone or in combination can be used as levers to controland/or manipulate glycosylation patterns while minimizing undesirableside effects, such as detrimental effects on cellular productivity.

The present invention is also applicable to modifying the glycosylationof a recombinant glycoprotein of interest such that it falls within thequality attribute ranges for the desired product. For example, thepresent invention is applicable to modifying the glycosylation of arecombinant glycoprotein of interest to more closely resemble, match, orsubstantially match the glycosylation pattern of a reference sample ofthe same glycoprotein. Differences between various manufacturingprocesses can result in glycoproteins with identical amino acidsequences having different glycosylation patterns depending on, forexample, conditions for growth, cell line used to express theglycoprotein, etc. Provided herein are methods for adjusting, altering,manipulating or changing the glycosylation pattern of a recombinantglycoprotein of interest comprising culturing eukaryotic cellsengineered to express the recombinant glycoprotein of interest in cellculture media that has been supplemented with an additive comprising oneor more of mycophenolic acid, mycophenolic acid acyl glucuronide,insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine,glucosamine, galactose, or mixtures thereof, or culturing eukaryoticcells engineered to express the recombinant glycoprotein of interest ina cell culture with a controlled or modulated (shifted) temperature, orculturing eukaryotic cells engineered to express the recombinantglycoprotein of interest in a cell culture with a controlled ormodulated seed density, or culturing eukaryotic cells engineered toexpress the recombinant glycoprotein of interest in cell culture with acontrolled or modulated (shifted) temperature and a cell culture mediathat has been supplemented with an additive comprising one or more ofmycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper(II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, galactose,or mixtures thereof, or culturing eukaryotic cells engineered to expressthe recombinant glycoprotein of interest in cell culture with acontrolled or modulated seed density and a cell culture media that hasbeen supplemented with an additive comprising one or more ofmycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper(II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, galactose,or mixtures thereof. According to the methods provided herein, any givencell culture can be adjusted using these components, either alone or incombination as levers to achieve or approach a desired glycosylationpattern while at the same time minimizing undesirable side effects.

I. Definitions

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. The terms “a” (or “an”), as well as theterms “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various embodimentsof the disclosure, which can be had by reference to the specification asa whole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The terms “polypeptide” or “protein” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. If a single polypeptide is thediscrete functioning unit and does require permanent physicalassociation with other polypeptides in order to form the discretefunctioning unit, the terms “polypeptide” and “protein” as used hereinare used interchangeably. If discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” as used herein refers to the multiple polypeptidesthat are physically coupled and function together as the discrete unit.

The term “glycoprotein” refers to a polypeptide or protein coupled to atleast one carbohydrate moiety, e.g., a polysaccharide or anoligosaccharide, that is attached to the protein via anoxygen-containing or a nitrogen-containing side chain of an amino acidresidue, e.g., a serine or threonine residue (“O-linked”) or anasparagine residue (“N-linked”). The term “glycan” refers to apolysaccharide or an oligosaccharide, e.g., a polymer comprised ofmonosaccharides. Glycans can be homo- or heteropolymers ofmonosaccharide residues, and can be linear or branched.

As used herein, the “glycosylation pattern” of a recombinantglycoprotein of interest refers to various physical characteristics ofthe glycoprotein's polysaccharides or oligosaccharides, such as, e.g.,the quantity and quality of various monosaccharides present, the degreeof branching, and/or the attachment (e.g., N-linked or O-linked). The“glycosylation pattern” of a glycoprotein can also refer to thefunctional characteristics imparted by the glycoprotein'soligosaccharides and polysaccharides. For example, the extent to whichthe glycoprotein can bind to FcγRIIIa and induce antibody-dependentcellular cytotoxicity (ADCC).

“Fucosylation” refers to the degree and distribution of fucose residueson polysaccharides and oligosaccharides, for example, N-glycans,0-glycans and glycolipids. Therapeutic glycoproteins, e.g., antibodiesor Fc fusion proteins, with non-fucosylated, or “afucosylated” N-glycansexhibit dramatically enhanced antibody-dependent cellular cytotoxicity(ADCC) due to the enhancement of FcγRIIIa binding capacity without anydetectable change in complement-dependent cytotoxicity (CDC) or antigenbinding capability. In certain situations, e.g., cancer treatment,non-fucosylated or “afucosylated” antibodies are desirable because theycan achieve therapeutic efficacy at low doses, while inducing highcellular cytotoxicity against tumor cells, and triggering high effectorfunction in NK cells via enhanced interaction with FcγRIIIa. In othersituations, e.g., treatment of inflammatory or autoimmune diseases,enhanced ADCC and FcγRIIIa binding is not desirable, and accordinglytherapeutic glycoproteins with higher levels of fucose residues in theirN-glycans can be preferable. As used herein, the term “% afucose” refersto the percentage of non-fucosylated N-glycans present on a recombinantglycoprotein of interest. A higher % afucose denotes a higher number ofnon-fucosylated N-glycans, and a lower % afucose denotes a higher numberof fucosylated N-glycans.

“Sialylation” refers to the type and distribution of sialic acidresidues on polysaccharides and oligosaccharides, for example,N-glycans, O-glycans and glycolipids. Sialic acids are most often foundat the terminal position of glycans. Sialylation can significantlyinfluence the safety and efficacy profiles of these proteins. Inparticular, the in vivo half-life of some biopharmaceuticals correlateswith the degree of oligosaccharide sialylation. Furthermore, thesialylation pattern can be a very useful measure of product consistencyduring manufacturing.

The two main types of sialyl residues found in biopharmaceuticalsproduced in mammalian expression systems are N-acetyl-neuraminic acid(NANA) and N-glycolylneuraminic acid (NGNA). These usually occur asterminal structures attached to galactose (Gal) residues at thenon-reducing terminii of both N- and O-linked glycans.

“Galactosylation” refers to the type and distribution of galactoseresidues on polysaccharides and oligosaccharides. Galactose refers to agroup of monosaccharides which include open chain and cyclic forms. Animportant disaccharide form of galactose isgalactose-alpha-1,3-galactose (α-gal).

The term “undesirable side effects” refers to certain aspects andresults of glycosylation which, under certain circumstances, are to beminimized or avoided. In certain aspects, a side effect to be reduced oravoided is a substantial increase in the level of α-gal. In anotheraspect a side effect to be reduced or avoided is a substantial reductionin sialic acid levels. In various aspects the methods described hereinachieve certain glycosylation patterns without substantially affectingculture density, cell viability level, or both. In certain aspects, a“side effect” which might be undesirable in one glycoprotein, e.g., andecrease in fucose levels (increases ADCC and FcγRIIIa binding) in anantibody used to treat an inflammatory disease, might be desirable inanother glycoprotein, e.g., in an antibody used to treat cancer.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Recombinantly expressed glycoprotein” and “recombinant glycoprotein” asused herein refer to a glycoprotein expressed from a host cell that hasbeen genetically engineered to express that glycoprotein. Therecombinantly expressed glycoprotein can be identical or similar toglycoproteins that are normally expressed in the mammalian host cell.The recombinantly expressed glycoprotein can also foreign to the hostcell, i.e. heterologous to peptides normally expressed in the mammalianhost cell. Alternatively, the recombinantly expressed glycoprotein canbe chimeric in that portions of the glycoprotein contain amino acidsequences that are identical or similar to glycoproteins normallyexpressed in the mammalian host cell, while other portions are foreignto the host cell. In certain embodiments, the recombinant glycoproteincomprises at least a portion of: an antibody, an immunoadhesin, aTransforming Growth Factor (TGF) beta superfamily signaling molecule, ablood clotting factor, combinations thereof, or fragments thereof. Asused herein, the terms “recombinantly expressed glycoprotein” and“recombinant glycoprotein” also encompasses an antibody produced by ahybridoma.

The term “expression” or “expresses” are used herein to refer totranscription and translation occurring within a host cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product gene that isproduced by the cell. For example, mRNA transcribed from a product geneis desirably quantitated by northern hybridization. Sambrook et al.,Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring HarborLaboratory Press, 1989). Protein encoded by a product gene can bequantitated either by assaying for the biological activity of theprotein or by employing assays that are independent of such activity,such as western blotting or radioimmunoassay using antibodies that arecapable of reacting with the protein. Sambrook et al., MolecularCloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring HarborLaboratory Press, 1989).

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 can be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of a moleculeof interest may be assessed in vivo, e.g., in an animal model such asthat disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native sourcethereof, e.g. from blood or PBMCs.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “basal media formulation” or “basal media” as used hereinrefers to any cell culture media used to culture cells that has not beenmodified either by supplementation, or by selective removal of a certaincomponent.

As used herein, the terms “additive” or “supplement” refer to anysupplementation made to a basal medium to achieve the goals described inthis disclosure. An “additive” or “supplement” can include a singlesubstance, e.g., mycophenolic acid, mycophenolic acid acyl glucuronide,insulin, copper II sulfate, or can include multiple substances, e.g.,copper II sulfate, hypoxanthine, and thymidine; mycophenolic acid andinsulin; mycophenolic acid and galactose; mycophenolic acid, galactose,and insulin; mycophenolic acid acyl glucuronide and insulin;mycophenolic acid acyl glucuronide and galactose; or mycophenolic acidacyl glucuronide, galactose, and insulin. The terms “additive” or“supplement” refer to the all of the components added, even though theyneed not be added at the same time, and they need not be added in thesame way. For example, one or more components of an “additive” or“supplement” can be added as a single bolus or two or more boli from astock solution, while other components of the same “additive” or“supplement” can be added as part of a feed medium. In addition, any oneor more components of an “additive” or “supplement” can be present inthe basal medium from the beginning of the cell culture.

The terms “culture”, “cell culture” and “eukaryotic cell culture” asused herein refer to a eukaryotic cell population, eithersurface-attached or in suspension that is maintained or grown in amedium (see definition of “medium” below) under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, these terms as used herein can refer tothe combination comprising the mammalian cell population and the mediumin which the population is suspended.

The terms “media”, “medium”, “cell culture medium”, “culture medium”,“tissue culture medium”, “tissue culture media”, and “growth medium” asused herein refer to a solution containing nutrients which nourishgrowing cultured eukaryotic cells. Typically, these solutions provideessential and non-essential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. The solution can also contain components that enhancegrowth and/or survival above the minimal rate, including hormones andgrowth factors. The solution is formulated to a pH and saltconcentration optimal for cell survival and proliferation. The mediumcan also be a “defined medium” or “chemically defined medium”—aserum-free medium that contains no proteins, hydrolysates or componentsof unknown composition. Defined media are free of animal-derivedcomponents and all components have a known chemical structure. One ofskill in the art understands a defined medium can comprise recombinantglycoproteins or proteins, for example, but not limited to, hormones,cytokines, interleukins and other signaling molecules.

The cell culture medium is generally “serum free” when the medium isessentially free of serum, or fractions thereof, from any mammaliansource (e.g. fetal bovine serum (FBS)). By “essentially free” is meantthat the cell culture medium comprises between about 0-5% serum,preferably between about 0-1% serum, and most preferably between about0-0.1% serum. Advantageously, serum-free “defined” medium can be used,wherein the identity and concentration of each of the components in themedium is known (i.e., an undefined component such as bovine pituitaryextract (BPE) is not present in the culture medium).

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. The term as used herein also refers to thatportion of cells which are alive at a particular time in relation to thetotal number of cells, living and dead, in the culture at that time.

The term “cell density” as used herein refers to that number of cellspresent in a given volume of medium.

The term “batch culture” as used herein refers to a method of culturingcells in which all the components that will ultimately be used inculturing the cells, including the medium (see definition of “medium”below) as well as the cells themselves, are provided at the beginning ofthe culturing process. A batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified.

The term “fed-batch culture” as used herein refers to a method ofculturing cells in which additional components are provided to theculture at some time subsequent to the beginning of the culture process.A fed-batch culture can be started using a basal medium. The culturemedium with which additional components are provided to the culture atsome time subsequent to the beginning of the culture process is a feedmedium. A fed-batch culture is typically stopped at some point and thecells and/or components in the medium are harvested and optionallypurified.

The term “perfusion culture” as used herein refers to a method ofculturing cells in which additional components are provided continuouslyor semi-continuously to the culture subsequent to the beginning of theculture process. The provided components typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. A portion of the cells and/or components in the medium aretypically harvested on a continuous or semi-continuous basis and areoptionally purified.

The term “bioreactor” as used herein refers to any vessel used for thegrowth of a mammalian cell culture. The bioreactor can be of any size solong as it is useful for the culturing of mammalian cells. Typically,the bioreactor will be at least 1 liter and can be 10, 50, 100, 250,500, 1000, 2000, 2500, 3000, 5000, 8000, 10,000, 12,0000, 15,000,20,000, 30,000 liters or more, or any volume in between. For example, abioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000liters, 10 to 20,000 liters, 10 to 30,000 liters, 50 to 5,000 liters, 50to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 50 to 30,000liters, 1,000 to 5,000 liters, or 1,000 to 3,000 liters. A bioreactorcan be a stirred-tank bioreactor or a shake flask. The internalconditions of the bioreactor, for example, but not limited to pH andtemperature, are typically controlled during the culturing period. Thebioreactor can be composed of any material that is suitable for holdingmammalian cell cultures suspended in media under the culture conditionsof the present invention, including glass, plastic or metal. The term“production bioreactor” as used herein refers to the final bioreactorused in the production of the glycoprotein or protein of interest. Thevolume of the large-scale cell culture production bioreactor istypically at least 500 liters and can be 1000, 2000, 2500, 5000, 8000,10,000, 12,0000, 15,000 liters or more, or any volume in between. Forexample, the large scale cell culture reactor will be between about 500liters and about 20,000 liters, about 500 liters and about 10,000liters, about 500 liters and about 5,000 liters, about 1,000 liters andabout 30,000 liters, about 2,000 liters and about 30,000 liters, about3,000 liters and about 30,000 liters, about 5,000 liters and about30,000 liters, or about 10,000 liters and about 30,000 liters, or alarge scale cell culture reactor will be at least about 500 liters, atleast about 1,000 liters, at least about 2,000 liters, at least about3,000 liters, at least about 5,000 liters, at least about 10,000 liters,at least about 15,000 liters, or at least about 20,000 liters. One ofordinary skill in the art will be aware of and will be able to choosesuitable bioreactors for use in practicing the present invention.

The term “stirred-tank bioreactor” as used herein refers to any vesselused for the growth of a mammalian cell culture that has an impeller.

The term “shake flask” as used herein refers to any vessel used for thegrowth of a mammalian cell culture that does not have an impeller.

The term “seeding” as used herein refers to the process of providing acell culture to a bioreactor or another vessel. In one embodiment, thecells have been propagated previously in another bioreactor or vessel.In another embodiment, the cells have been frozen and thawed immediatelyprior to providing them to the bioreactor or vessel. The term refers toany number of cells, including a single cell.

“Growth phase” of the cell culture refers to the period of exponentialcell growth (the log phase) where cells are generally rapidly dividing.During this phase, cells are cultured for a period of time, usuallybetween 1-4 days, and under such conditions that cell growth ismaximized. The determination of the growth cycle for the host cell canbe determined for the particular host cell envisioned without undueexperimentation. “Period of time and under such conditions that cellgrowth is maximized” and the like, refer to those culture conditionsthat, for a particular cell line, are determined to be optimal for cellgrowth and division. During the growth phase, cells are cultured innutrient medium containing the necessary additives generally at about25°−40° C., in a humidified, controlled atmosphere, such that optimalgrowth is achieved for the particular cell line. Cells are maintained inthe growth phase for a period of about between one and seven days, e.g.,between two to six days, e.g., six days. The length of the growth phasefor the particular cells can be determined without undueexperimentation. For example, the length of the growth phase will be theperiod of time sufficient to allow the particular cells to reproduce toa viable cell density within a range of about 20%-80% of the maximalpossible viable cell density if the culture was maintained under thegrowth conditions.

“Production phase” or “protein production phase” of the cell culturerefers to the period of time during which cell growth has plateaued.During the production phase, logarithmic cell growth has ended andprotein production is primary. During this period of time the medium isgenerally supplemented to support continued protein production and toachieve the desired glycoprotein product. The production phase istypically between about three and about ten days, e.g., between aboutfive and about eight days, e.g., six days.

The term “hybridoma” as used herein refers to a cell created by fusionof an immortalized cell derived from an immunologic source and anantibody-producing cell. The resulting hybridoma is an immortalized cellthat produces antibodies. The individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. The term alsoencompasses trioma cell lines, which result when progeny of heterohybridmyeloma fusions, which are the product of a fusion between human cellsand a murine myeloma cell line, are subsequently fused with a plasmacell. Furthermore, the term is meant to include any immortalized hybridcell line that produces antibodies such as, for example, quadromas (See,e.g., Milstein et al., Nature, 537:3053 (1983)).

The term “osmolality” is a measure of the osmotic pressure of dissolvedsolute particles in an aqueous solution. The solute particles includeboth ions and non-ionized molecules. Osmolality is expressed as theconcentration of osmotically active particles (i.e., osmoles) dissolvedin 1 kg of water (1 mOsm/kg H₂O at 38° C. is equivalent to an osmoticpressure of 19 mm Hg). “Osmolarity” refers to the number of soluteparticles dissolved in 1 liter of solution. Solutes which can be addedto the culture medium so as to increase the osmolality thereof includeproteins, peptides, amino acids, non-metabolized polymers, vitamins,ions, salts, sugars, metabolites, organic acids, lipids, etc. In thepreferred embodiment, the concentration of amino acids and NaCl in theculture medium is increased in order to achieve the desired osmolalityranges set forth herein. When used herein, the abbreviation “mOsm” means“milliosmoles/kg H₂O”.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed glycoprotein or protein produced by a cellculture divided by a given amount of medium volume. Titer is typicallyexpressed in units of milligrams of glycoprotein or protein permilliliter of medium or in units of grams of glycoprotein or protein perliter of medium.

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a CH2/CH3 region, such as an Fc region. Protein Acan be purchased commercially from Repligen, Pharmacia and Fermatech.Protein A is generally immobilized on a solid phase support material.The term “ProA” also refers to an affinity chromatography resin orcolumn containing chromatographic solid support matrix to which iscovalently attached Protein A.

The term “chromatography” refers to the process by which a solute ofinterest in a mixture is separated from other solutes in a mixture as aresult of differences in rates at which the individual solutes of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably herein and refer to a protein separationtechnique in which a protein of interest or antibody of interest isreversibly and specifically bound to a biospecific ligand. Preferably,the biospecific ligand is covalently attached to a chromatographic solidphase material and is accessible to the protein of interest in solutionas the solution contacts the chromatographic solid phase material. Theprotein of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein of interest is then removed inactive form from the immobilized ligand with low pH, high pH, high salt,competing ligand, and the like, and passed through the chromatographiccolumn with the elution buffer, free of the contaminating proteins orprotein impurities that were earlier allowed to pass through the column.Any component can be used as a ligand for purifying its respectivespecific binding protein, e.g. antibody.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification process in which affinity chromatography is notutilized. Non-affinity chromatography includes chromatographictechniques that rely on non-specific interactions between a molecule ofinterest (such as a protein, e.g. antibody) and a solid phase matrix.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, sulphopropyl(SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™ orSP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from Pharmacia). A“mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties. Acommercially available mixed mode ion exchange resin is BAKERBOND ABX™(J.T. Baker, Phillipsburg, N.J.) containing weak cation exchange groups,a low concentration of anion exchange groups, and hydrophobic ligandsattached to a silica gel solid phase support matrix.

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 2 to about 9,alternatively from about 3 to about 8, alternatively from about 4 toabout 7 alternatively from about 5 to about 7. Non-limiting examples ofbuffers that will control the pH in this range include IVIES, MOPS,MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammoniumbuffers, as well as combinations of these.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., cellular viability). Thedifference between said two values is, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein with regard to amounts or numerical values (and not asreference to the chemical process of reduction), denotes a sufficientlyhigh degree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., cellular viability). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

II. Supplementation of Cell Culture Medium to Alter GlycosylationPatterns

Provided herein are methods to culture eukaryotic cells engineered toexpress a recombinant glycoprotein of interest. Specifically thisdisclosure provides methods for altering the glycosylation patterns of arecombinant glycoprotein of interest by supplementing a tissue culturemedium in which the cells are growing and/or producing the recombinantglycoprotein of interest with an additive, or culturing eukaryotic cellsengineered to express a glycoprotein of interest in a tissue culturemedium which has been supplemented with such an additive. In certainembodiments, glycoproteins produced by the methods provided arerecovered. The methods are based on the recognition that growth of cellsexpressing a recombinant glycoprotein of interest in cell culture mediumsupplemented with an additive comprising one or more of mycophenolicacid, mycophenolic acid acyl glucuronide, insulin, copper (II) sulfate,hypoxanthine, thymidine, guanine, glucosamine (GlcN), galactose ormixtures thereof can result in alterations to eukaryotic cellglycosylation patterns, such as the level of afucosylation,galactosylation, N-glycan charge, N-glycolylneuraminic acid (NGNA), andFcγRIIIa binding. In certain embodiments, the alteration of theglycosylation pattern of the recombinant glycoprotein of interestcomprises one or more of a reduced level of afucosylation, a reducedlevel of galactosylation, a reduced level ofgalactose-alpha-1,3-galactose (α-gal), a reduced level ofN-glycolylneuraminic acid (NGNA), reduced FcγRIIIa binding, reducedantibody-dependent cell-mediated cytotoxicity, or an increased N-glycancharge. In certain embodiments, the alteration of the glycosylationpattern of the recombinant glycoprotein of interest comprises a reducedlevel of afucosylation. In certain embodiments, the alteration of theglycosylation pattern of the recombinant glycoprotein of interestcomprises a increased level of afucosylation.

As the additives described herein have differential effects on bothglycosylation and culture conditions, addition of a given additive canbe used to control and/or manipulate glycosylation patterns whileminimizing one or more undesirable side effects, e.g., side effectsaffecting cellular productivity. For example, in certain embodiments adesirable glycosylation pattern is achieved (e.g., a reduced percentageof afucosylated N-glycans) without substantially increasing the levelsof α-gal, which in some instances can be an undesirable side effect. Inother embodiments, desirable glycosylation patterns are achieved withoutsubstantially reducing sialic acid levels, which can be an undesirableside effect. The desirability of each additive or additive combinationdepends on the application. If a product quality attribute isprioritized over another product quality attribute affected by the sideeffect of an additive or additive combination, then providing theadditive or additive combination would still be desirable.

For example, as discussed in more detail below, if reducingafucosylation is a priority and cell titer is less important, then anadditive comprising copper (II) sulfate plus hypoxanthine can bedesirable because it can generate a dramatic effect on afucosylation.However, if titer is of greatest importance, then a more desirablesolution becomes an additive comprising copper (II) sulfate withouthypoxanthine, because some reduction in afucosylation is achievedwithout substantially affecting cell titer. Further, if increasingafucosylation is of a greater importance and cell viability, viable celldensity, and titer are of less importance, then an additive comprisingmycophenolic acid or mycophenolic acid acyl glucuronide can bedesirable. However, if cell viability and viable cell density and titerare also important, then an additive comprising mycophenolic acid andinsulin can be desirable. In other words, the various possible additivecomponents can be used as levers to achieve the most desirable balancebetween approaching a particular glycosylation pattern and minimizingside effects.

The present invention is also applicable to altering, manipulating, orcontrolling the glycosylation pattern of a recombinant glycoprotein ofinterest to match, substantially match, approach, or more closelyresemble the glycosylation pattern of the same glycoprotein, butproduced in a different cell culture system. Recombinant glycoproteinsof interest can be produced according to the invention using variousdifferent cell culture systems, e.g., a batch culture, fed-batch culturea perfusion culture, a shake flask, and/or a bioreactor. In oneembodiment, cells expressing a recombinant glycoprotein of interest arecultured in basal medium to which the additive is introduced as a bolus,or two or more boli, from a stock solution. In another embodiment, theadditive is introduced as a component of a feed medium. In certainembodiments the cell culture comprises a growth phase and a proteinproduction phase, and the additive is introduced into the culture mediumbefore, or at the same time as, or at some point after the initiation ofthe protein production phase.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

In certain embodiments, the additive comprises glucosamine (GlcN). Incertain related embodiments the additive comprises GlcN plus galactose.An additive comprising GlcN or GlcN plus galactose can be used in aculture medium, for example, to reduce the level of afucosylation (e.g.,maintain sufficient levels of fucosylated N-glycans), to reduce thelevels α-gal, or to reduce the levels of NGNA of a recombinantglycoprotein of interest. In certain embodiments, the use of an additivecomprising GlcN or GlcN plus galactose must be balanced with possibleside effects, e.g., a slight reduction in cell density and viability, areduced level of total sialic acid, or the appearance of unknownsialylated species.

In certain embodiments the additive comprises GlcN, which can be addedto the culture medium in one bolus or two or more boli from a stocksolution to achieve a GlcN concentration in the culture medium ofbetween about 1 mM and about 100 mM. For example sufficient GlcN isadded to achieve a GlcN concentration in the culture medium of betweenabout 1 mM and about 90 mM, about 1 mM and about 80 mM, about 1 mM andabout 70 mM, about 1 mM and about 60 mM, about 5 mM and about 50 mM,about 5 mM and about 40 mM, about 5 mM and about 30 mM, about 5 mM andabout 20 mM, or about 5 mM and about 10 mM. In certain embodiments GlcNis added to achieve a GlcN concentration in the culture medium of about10 mM, added as a single 10 mM bolus, or about 20 mM added as two 10 mMboli, either on the same day or on separate days. In certain embodimentsthe additive comprises GlcN, e.g., at the concentrations listed above,and further comprises galactose, which can be added as a component of afeed medium, to achieve a galactose concentration in the culture mediumof between about 1 g/L to about 50 g/L, for example, between about 1 g/Land about 40 g/L, about 1 g/L and about 30 g/L, about 1 g/L and about 20g/L, about 1 g/L and about 10 g/L, or about 2 g/L and about 6 g/L. Incertain embodiments galactose is added to achieve a final concentrationin the cell culture medium of about 2 g/L, about 3 g/L, about 4 g/L,about 5 g/L, or about 6 g/L. In certain embodiments the additivecomprises GlcN, added to achieve a final concentration of about 10 mM orabout 20 mM and galactose, added to achieve a final concentration ofabout 2 g/L, about 4 g/L, or about 6 g/L.

In certain embodiments, the additive comprises mycophenolic acid (MPA).Mycophenolic acid is known to inhibit the enzyme inosine monophosphatedehydrogenase (IMPDH) that is involved in the synthesis of guaninenucleotides (GMP) (Huang et al., Leukemia Research 32:131-141 (2008))and to cause alterations in the formation of endothelial or surfaceglycoproteins (Bertalanffy et al., Clin. Chem. Lab. Med., 37(3):259-264(1999)). The inventors have found that MPA increases the level ofafucosylation of a recombinant protein produced in cell culture.

In certain related embodiments, the additive comprises MPA plus insulin,or MPA plus galactose, or MPA plus insulin and galatose. An additivecomprising MPA or MPA plus galactose can be used in a culture medium,for example, to increase the level of afucosylation. In certainembodiments, the use of an additive comprising MPA or MPA plus galactosemust be balanced with possible side effects, e.g., a reduction in celldensity and viability, a reduced titer, or a shift in the glycan ratios.

As the supplementation of cell culture medium with mycophenolic acid hasdifferential effects on both glycosylation and culture conditions,mycophenolic acid can be used to control and/or manipulate glycosylationpatterns. In certain embodiments, the mycophenolic acid is used as asupplement in the cell culture medium to increase afucosylation level ofthe recombinant protein.

In certain embodiments the additive comprises MPA, which can be added tothe culture medium in one bolus or two or more boli from a stocksolution to achieve a MPA concentration in the culture medium of betweenabout 1 μM and about 50 μM. For example sufficient MPA is added toachieve a MPA concentration in the culture medium of between about 1 μMand about 45 μM, about 1 μM and about 40 μM, about 1 μM and about 30 μM,about 1 μM and about 25 μM, about 5 μM and about 40 μM, about 5 andabout 35 μM, about 5 μM and about 30 μM, about 5 μM and about 25 μM, orabout 5 and about 20 μM. In certain embodiments MPA is added to achievea MPA concentration in the culture medium of about 1 μM, about 2.5 μM,about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 48 μM,or about 50 μM. In certain embodiments, MPA is added as a single bolusor as two or more boli, either on the same day or on separate days. Incertain embodiments the additive comprises MPA, e.g., at theconcentrations listed above, and further comprises galactose, which canbe added as a component of a feed medium, to achieve a galactoseconcentration in the culture medium of between about 1 g/L to about 50g/L, for example, between about 1 g/L and about 40 g/L, about 1 g/L andabout 30 g/L, about 1 g/L and about 20 g/L, about 1 g/L and about 10g/L, or about 1 g/L and about 5 g/L, or about or about 2 g/L and about50 g/L or 2 g/L and about 40 g/L, or about 2 g/L and about 30 g/L, orabout 2 g/L and about 20 g/L, about 2 g/L and about 10 g/L, or 5 g/L andabout 50 g/L, or about 5 g/L and about 40 g/L, or about 5 g/L and about30 g/L, or about 5 g/L and about 20 g/L, or about 5 g/L and about 10g/L. In certain embodiments galactose is added to achieve a finalconcentration in the cell culture medium of about 2 g/L, about 3 g/L,about 4 g/L, about 5 g/L, or about 6 g/L, or about 10 g/L, or about 15g/L, or about 20 g/L, or about 25 g/L, or about 30 g/L, or about 35 g/L,or about 40 g/L, or about 45 g/L, or about 50 g/L.

In certain embodiments, the additive comprises mycophenolic acid acylglucuronide (acMPAG). Mycophenolic acid acyl glucuronide is aglucuronidation metabolite of mycophenolic acid. The inventors havefound that acMPAG increases the level of afucosylation of a recombinantprotein produced in cell culture.

In certain related embodiments, the additive comprises acMPAG plusinsulin, or acMPAG plus galactose, or acMPAG plus insulin and galatose.An additive comprising acMPAG or acMPAG plus galactose can be used in aculture medium, for example, to increase the level of afucosylation. Incertain embodiments, the use of an additive comprising acMPAG or acMPAGplus galactose must be balanced with possible side effects, e.g., areduction in cell density and viability, a reduced titer, or a shift inthe glycan ratios.

As the supplementation of cell culture medium with mycophenolic acidacyl glucuronide has differential effects on both glycosylation andculture conditions, mycophenolic acid acyl glucuronide can be used tocontrol and/or manipulate glycosylation patterns. In certainembodiments, the mycophenolic acid acyl glucuronide is used as asupplement in the cell culture medium to increase afucosylation level ofthe recombinant protein.

In certain embodiments the additive comprises acMPAG, which can be addedto the culture medium in one bolus or two or more boli from a stocksolution to achieve acMPAG concentration in the culture medium ofbetween about 1 μM and about 50 μM. For example sufficient acMPAG isadded to achieve acMPAG concentration in the culture medium of betweenabout 1 μM and about 45 μM, about 1 μM and about 40 μM, about 1 μM andabout 30 μM, about 1 μM and about 25 μM, about 5 μM and about 40 μM,about 5 μM and about 35 μM, about 5 μM and about 30 μM, about 5 μM andabout 25 μM, or about 5 μM and about 20 μM. In certain embodimentsacMPAG is added to achieve acMPAG concentration in the culture medium ofabout 1 μM, about 2.5 μM, about 5 μM, about 7.5 μM, about 10 μM, about15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM,about 45 μM, about 48 μM, or about 50 μM. In certain embodiments, acMPAGis added as a single bolus or as two or more boli, either on the sameday or on separate days. In certain embodiments the additive comprisesacMPAG, e.g., at the concentrations listed above, and further comprisesgalactose, which can be added as a component of a feed medium, toachieve a galactose concentration in the culture medium of between about1 g/L to about 50 g/L, for example, between about 1 g/L and about 40g/L, about 1 g/L and about 30 g/L, about 1 g/L and about 20 g/L, about 1g/L and about 10 g/L, or about 1 g/L and about 5 g/L, or about or about2 g/L and about 50 g/L or 2 g/L and about 40 g/L, or about 2 g/L andabout 30 g/L, or about 2 g/L and about 20 g/L, about 2 g/L and about 10g/L, or 5 g/L and about 50 g/L, or about 5 g/L and about 40 g/L, orabout 5 g/L and about 30 g/L, or about 5 g/L and about 20 g/L, or about5 g/L and about 10 g/L. In certain embodiments galactose is added toachieve a final concentration in the cell culture medium of about 2 g/L,about 3 g/L, about 4 g/L, about 5 g/L, or about 6 g/L, or about 10 g/L,or about 15 g/L, or about 20 g/L, or about 25 g/L, or about 30 g/L, orabout 35 g/L, or about 40 g/L, or about 45 g/L, or about 50 g/L.

In certain embodiments, the additive comprises insulin. Insulin is knownto stimulate the transport and phosphorylation of pyrimidineribonucleotides in isolated bone cells (Peck et al, J. Biol. Chem.,245(10):2722-2729 (1970)). The inventors have found that insulinincreases afucosylation level of a recombinant glycoprotein produced incell culture.

In certain related embodiments the additive comprises insulin plusmycophenolic acid (MPA) and/or mycophenolic acid acyl glucuronide(acMPAG). An additive comprising insulin or insulin plus MPA and/oracMPAG can be used in a culture medium, for example, to increase thelevel of afucosylation (e.g., maintain sufficient levels of fucosylatedN-glycans). In certain embodiments, the use of an additive comprisinginsulin or insulin plus MPA and/or acMPAG must be balanced with possibleside effects, e.g., a slight reduction in cell density and viability.

As the supplementation of cell culture medium with insulin hasdifferential effects on both glycosylation and culture conditions,insulin can be used to control and/or manipulate glycosylation patternswithout having any undesirable side effects, e.g., side effectsaffecting cellular productivity. In certain embodiments, the insulin isused as a supplement in the cell culture medium to increaseafucosylation level of the recombinant protein.

In certain embodiments the additive comprises insulin, which can beadded to the culture medium in one bolus or two or more boli from astock solution to achieve an insulin concentration in the culture mediumof between about 1 mg/L and about 50 mg/L. For example sufficientinsulin is added to achieve an insulin concentration in the culturemedium of between about 1 mg/L and about 40 mg/L, or 1 mg/L and about 30mg/L, or about 1 mg/L and about 25 mg/L, 1 mg/L and about 22.5 mg/L, orabout 1 mg/L and about 20 mg/L, or 1 mg/L and about 15 mg/L, or about 1mg/L and about 15 mg/L, or about 1 mg/L and about 10 mg/L, or about 5mg/L and about 50 mg/L, or about 5 mg/L and about 40 mg/L, or about 5mg/L and about 30 mg/L, or about 5 mg/L and about 25 mg/L, or about 5mg/L and about 20 mg/L, or about 5 mg/L and about 15 mg/L, or about 5mg/L and about 10 mg/L, or about 10 mg/L and about 50 mg/L, or about 10mg/L and about 40 mg/L, or about 10 mg/L and about 30 mg/L, or about 10mg/L and about 25 mg/L, or about 10 mg/L and about 20 mg/L, or about 10mg/L and about 15 mg/L, or about 15 mg/L and about 25 mg/L, or about 15mg/L and about 20 mg/L, or about 17 mg/L and about 25 mg/L, or about 17mg/L and about 20 mg/L, or about 17 mg/L and about 24 mg/L. In certainembodiments insulin is added to achieve an insulin concentration in theculture medium of about 10.6 mg/L, about 13 mg/L, about 17.2 mg/L, orabout 24.4 mg/L added as a single bolus, or as two or more boli, eitheron the same day or on separate days. In certain embodiments the additivecomprises insulin, e.g., at the concentrations listed above, and furthercomprises mycophenolic acid (MPA) and/or mycophenolic acid acylglucuronide (acMPAG), which can be added as a component of a feedmedium, to achieve a MPA and/or acMPAG concentration in the culturemedium of between about 1 μM and about 50 μM, for example, between about1 μM and about 45 μM, about 1 μM and about 40 μM, about 1 μM and about30 μM, about 1 μM and about 25 μM, about 5 μM and about 40 μM, about 5μM and about 35 μM, about 5 μM and about 30 μM, about 5 μM and about 25μM, or about 5 μM and about 20 μM. In certain embodiments MPA and/oracMPAG is added to achieve a final concentration in the cell culturemedium of about 1 μM, about 2.5 μM, about 5 μM, about 7.5 μM, about 10μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM,about 40 μM, about 45 μM, about 48 μM, or about 50 μM. In certainembodiments the additive comprises insulin, added to achieve a finalconcentration of about 10.6 mg/L, about 13 mg/L, about 17.2 mg/L, orabout 24.4 mg/L and galactose, added to achieve a final concentration ofabout 2 g/L, about 4 g/L, or about 6 g/L, or about 10 g/L.

In certain embodiments, the additive comprises copper (II) sulfate(CuSO₄). In certain embodiments the additive comprises CuSO₄ and furthercomprises GlcN and/or galactose. In certain embodiments the additivecomprises CuSO₄ and further comprises hypoxanthine. In certainembodiments the additive comprises CuSO₄ and hypoxanthine, and furthercomprises thymidine. An additive comprising CuSO₄, CuSO₄, GlcN, andgalactose, CuSO₄ and hypoxanthine, or CuSO₄, hypoxanthine, andthymidine, can be used in a culture medium, for example, to reduce thelevel of afucosylation (e.g., maintain sufficient levels of fucosylatedN-glycans), to reduce the level of galactosylation, and/or to increasethe level of charged N-glycans of a recombinant glycoprotein ofinterest. In certain embodiments, the use of an additive comprisingCuSO₄, CuSO₄, GlcN, and galactose, CuSO₄ and hypoxanthine, or CuSO₄,hypoxanthine, and thymidine must be balanced with possible side effects,e.g., a slight reduction in cell density and viability, or a reducedlevel of total sialic acid.

In certain embodiments the additive comprises CuSO₄, which can be addedto the culture medium in one bolus or two or more boli from a stocksolution to, or be added as a component of a feed medium achieve a CuSO₄concentration in the culture medium of between about 0.05 mM and about10 mM CuSO₄. In certain embodiments the additive comprises CuSO₄, whichcan be added to the culture medium in one bolus or two or more boli froma stock solution to, or be added as a component of a feed medium achievea CuSO₄ concentration in the culture medium between about 0.1 mM andabout 10 mM, about 0.2 mM and about 5 mM, about 0.2 mM and about 4 mM,about 0.2 mM and about 3 mM, about 0.2 mM and about 2 mM, about 0.2 mMand about 1 mM, or about 0.2 mM and about 0.5 mM.

In certain embodiments the additive comprises CuSO₄, e.g., at theconcentrations listed above, and further comprises GlcN and galactose,which can be added as one bolus or two or more boli of a stock solutionor as a component of a feed medium to achieve GlcN concentrationsbetween about 1 mM and about 100 mM, about 1 mM and about 90 mM, about 1mM and about 80 mM, about 1 mM and about 70 mM, about 1 mM and about 60mM, about 5 mM and about 50 mM, about 5 mM and about 40 mM, about 5 mMand about 30 mM, about 5 mM and about 20 mM, or about 5 mM and about 10mM; and a galactose concentration of between about 1 g/L to about 50g/L, for example, between about 1 g/L and about 40 g/L, about 1 g/L andabout 30 g/L, about 1 g/L and about 20 g/L, about 1 g/L and about 10g/L, or about 2 g/L and about 6 g/L. In certain embodiments galactose isadded to achieve a final concentration in the cell culture medium ofabout 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, or about 6 g/L. Incertain embodiments the additive comprises GlcN, added to achieve afinal concentration of about 10 mM or about 20 mM and galactose, addedto achieve a final concentration of about 2 g/L, about 4 g/L, or about 6g/L. In certain embodiments the additive comprises CuSO₄, e.g., at about0.2 mM or about 0.5 mM, and further comprises GlcN and galactose toachieve a GlcN concentration in the culture medium of about 10 mM orabout 20 mM and a galactose concentration of about 2 g/L, about 4 g/L,or about 6 g/L.

In certain embodiments the additive comprises CuSO₄, e.g., at theconcentrations listed above, and further comprises hypoxanthine, whichcan be added either as a single bolus or two or more boli from a stocksolution or can be added as a component of a feed medium, to achieve ahypoxanthine concentration of between about 0.1 mM and about 10 mM,about 0.3 mM and about 5 mM, about 0.3 mM and about 4 mM, about 0.3 mMand about 3 mM, about 0.3 mM and about 2 mM, about 0.3 mM and to about 1mM, about 0.5 mM and about 0.9 mM, about 0.5 mM and about 0.8 mM, orabout 0.5 mM and about 0.7 mM. In certain embodiments, the additivecomprises CuSO₄ and hypoxanthine to achieve a final concentration in thecell culture medium of about 0.23 mM CuSO₄ and about 0.7 mMhypoxanthine.

In certain embodiments the additive comprises CuSO₄ and hypoxanthine,e.g., at the concentrations listed above, and further comprisesthymidine, which can be added as a component of a feed medium or as asingle bolus or two or more boli from a stock solution, to achieve afinal concentration in the cell culture medium of between about 0.005 mMand about 5 mM, about 0.005 mM and about 1 mM about 0.005 mM and about0.5 mM, about 0.01 mM and about 0.1 mM, about 0.01 mM and about 0.05 mM,about 0.01 mM and about 0.2 mM, or about 0.05 mM and about 0.2 mM. Incertain embodiments, the additive comprises CuSO₄, hypoxanthine, andthymidine to achieve final concentrations in the cell culture medium ofabout 0.3 mM CuSO₄, about 0.3 mM hypoxanthine, and about 0.05 mMthymidine, or about 0.3 mM CuSO₄, about 1 mM hypoxanthine, and about0.16 mM thymidine.

In certain embodiments, the additive comprises hypoxanthine, which canbe added either as a single bolus or two or more boli from a stocksolution or can be added as a component of a feed medium, to achieve ahypoxanthine concentration in the cell culture medium of between about0.1 mM and about 10 mM, about 0.3 mM and about 5 mM, about 0.3 mM andabout 4 mM, about 0.3 mM and about 3 mM, about 0.3 mM and about 2 mM,about 0.3 mM and to about 1 mM, about 0.5 mM and about 0.9 mM, about 0.5mM and about 0.8 mM, or about 0.5 mM and about 0.7 mM. In certainembodiments the additive comprises hypoxanthine to achieve ahypoxanthine concentration in the cell culture medium of about 1 mM. Incertain embodiments the additive comprises guanine, which can be addedeither as a single bolus or two or more boli from a stock solution orcan be added as a component of a feed medium, to achieve a guanineconcentration in the cell culture medium of between about 0.1 mM andabout 10 mM, about 0.3 mM and about 5 mM, about 0.3 mM and about 4 mM,about 0.3 mM and about 3 mM, about 0.3 mM and about 2 mM, about 0.3 mMand to about 1 mM, about 0.5 mM and about 0.9 mM, about 0.5 mM and about0.8 mM, or about 0.5 mM and about 0.7 mM. In certain embodiments theadditive comprises guanine to achieve a guanine concentration in thecell culture medium of about 0.2 mM. An additive comprising hypoxanthineor guanine can be used in a culture medium, for example, to reduce thelevel of afucosylation (e.g., maintain sufficient levels of fucosylatedN-glycans) or to reduce the levels of FcγRIIIa binding. In certainembodiments, the use of an additive comprising hypoxanthine or guaninemust be balanced with possible side effects, e.g., a slight reduction incell density and viability.

III. Control or Modulation of Cell Culture Temperature to AlterGlycosylation Pattern

Provided herein are methods to culture eukaryotic cells engineered toexpress a recombinant glycoprotein of interest. Specifically, thisdisclosure provides methods for altering the glycosylation patterns of arecombinant glycoprotein of interest by controlling or modulating(shifting) temperature of a cell culture in which the cells are growingand/or producing the recombinant glycoprotein of interest or culturingeukaryotic cells engineered to express a glycoprotein of interest in acell culture that has a controlled or modulated (shifted) temperature.

In certain embodiments, glycoproteins produced by the methods providedare recovered. The methods are based on the recognition that growth ofcells expressing a recombinant glycoprotein of interest in cell culturemedium with a controlled or modulated (shifted) temperature can resultin alterations to eukaryotic cell glycosylation patterns, such as thelevel of afucosylation, galactosylation, N-glycan charge,N-glycolylneuraminic acid (NGNA), and FcγRIIIa binding. In certainembodiments, the alteration of the glycosylation pattern of therecombinant glycoprotein of interest comprises one or more of a reducedlevel of afucosylation, a reduced level of galactosylation, a reducedlevel of galactose-alpha-1,3-galactose (α-gal), a reduced level ofN-glycolylneuraminic acid (NGNA), reduced FcγRIIIa binding, reducedantibody-dependent cell-mediated cytotoxicity, or an increased N-glycancharge. In certain embodiments, the alteration of the glycosylationpattern of the recombinant glycoprotein of interest comprises anincreased level of afucosylation.

As the temperature of the cell culture has differential effects on bothglycosylation and culture conditions, temperature can be used to controlor modulate (shift) glycosylation patterns while minimizing one or moreundesirable side effects, e.g., side effects affecting cellularproductivity. In certain embodiments, the control or modulation of cellculture temperature comprises a reduction in the temperature to resultin an increased levels of afucosylation. In certain embodiments, themodulation of cell culture temperature comprises a reduction in thetemperature to result in a decreased levels of afucosylation. In otherembodiments, the control or modulation of cell culture temperaturecomprises an increase in the temperature to result in an increasedlevels of afucosylation. In certain embodiments, the modulation of cellculture temperature comprises an increase in the temperature to resultin a decreased levels of afucosylation.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bysupplementing a culture medium in which the cells are growing with anadditive and controlling or modulating (shifting) the temperature of thecell culture. In certain embodiments, the disclosure provides methodsfor producing the recombinant glycoprotein of interest with an additivein a culture medium that has controlled or modulated (shifted) cellculture temperature, or culturing eukaryotic cells engineered to expressa glycoprotein of interest in a tissue culture medium that has beensupplemented with an additive and has a controlled or modulated(shifted) temperature. For example, in certain embodiments a desirableglycosylation pattern is achieved (e.g., a reduced percentage ofafucosylated N-glycans) without substantially increasing the levels ofα-gal, which in some instances can be an undesirable side effect. Inother embodiments, desirable glycosylation patterns are achieved withoutsubstantially reducing sialic acid levels, which can be an undesirableside effect. The desirability of each combination depends on theapplication. If a product quality attribute is prioritized over theproduct quality attribute affected by the corresponding side effect,then providing the additive would still be desirable.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bysupplementing the medium in which the cells are growing withmycophenolic acid as an additive and modifying the cell culturetemperature. In certain embodiments, the disclosure provides methods foraltering the glycosylation patterns of a recombinant glycoprotein ofinterest by supplementing the medium in which the cells are growing withmycophenolic acid acyl glucuronide as an additive and modifying the cellculture temperature. In certain embodiments, the disclosure providesmethods for producing the recombinant glycoprotein of interest withmycophenolic acid as an additive in a culture medium that has controlledor manipulated cell culture temperature, or culturing eukaryotic cellsengineered to express a glycoprotein of interest in a tissue culturemedium that has been supplemented with mycophenolic acid as an additiveand has a controlled or modulated (shifted) temperature. In certainembodiments, the disclosure provides methods for producing therecombinant glycoprotein of interest with mycophenolic acid acylglucuronide as an additive in a culture medium that has controlled ormanipulated cell culture temperature, or culturing eukaryotic cellsengineered to express a glycoprotein of interest in a tissue culturemedium that has been supplemented with mycophenolic acid acylglucuronide as an additive and has a controlled or modulated (shifted)temperature.

In certain embodiments, controlling or modulating (shifting) thetemperature of the cell culture plus supplementing the culture mediumwith an additive comprising mycophenolic acid can be desirable. Incertain embodiments, controlling or modulating (shifting) thetemperature of the cell culture plus supplementing the culture mediumwith an additive comprising mycophenolic acid acyl glucuronide can bedesirable. In certain embodiments, controlling or modulating (shifting)the temperature of the cell culture plus supplementing the culturemedium with an additive comprising insulin can be desirable. In otherwords, the various possible additive components can be used as levers toachieve the most desirable balance between approaching a particularglycosylation pattern and minimizing side effects. The present inventionis also applicable to altering, manipulating, or controlling theglycosylation pattern of a recombinant glycoprotein of interest tomatch, substantially match, approach, or more closely resemble theglycosylation pattern of the same glycoprotein, but produced in adifferent cell culture system. Recombinant glycoproteins of interest canbe produced according to the invention using various different cellculture systems, e.g., a batch culture, fed-batch culture a perfusionculture, a shake flask, and/or a bioreactor. In one embodiment, cellsexpressing a recombinant glycoprotein of interest are cultured in basalmedium to which the additive is introduced as a bolus, or two or moreboli, from a stock solution. In another embodiment, the additive isintroduced as a component of a feed medium.

In certain embodiments the cell culture comprises a growth phase and aprotein production phase, and the cell culture temperature is controlledor modulated (shifted) before, or at the same time as, or at some pointafter the initiation of the protein production phase. In certainembodiments, the additive is introduced into the culture medium before,or at the same time as, or at some point after the initiation of theprotein production phase. In certain embodiments, the additive isintroduced and the temperature is controlled or modulated (shifted) atthe same time. In certain embodiments, the additive is introduced andthe cell culture temperature is controlled or modulated (shifted) atdifferent times.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

In certain embodiments, the controlling or modulating (shifting) of cellculture temperature comprises decreasing the culture temperature up to25° C. In certain embodiments, the controlling or modulating (shifting)of cell culture temperature comprises increasing the culture temperatureup to 42° C. In certain embodiments, the cell culture temperature iscontrolled or modulated (shifted) to about 25° C., about 25.5° C., about26° C., about 26.5° C., about 27° C., about 27.5° C., about 28° C.,about 28.5° C., about 29° C., about 29.5° C., about 30° C., about 30.5°C., about 31° C., about 31.5° C., about 32° C., about 32.5° C., about33° C., about 33.5° C., about 34° C., about 34.5° C., about 35° C.,about 35.5° C., about 36° C., about 36.5° C., about 37° C., about 37.5°C., about 38° C., about 38.5° C., about 39° C., about 39.5° C., about40° C., about 40.5° C., about 41° C., about 41.5° C., or about 42° C.

IV. Control or Modulation of Seed Density to Alter Glycosylation Pattern

Provided herein are methods to culture eukaryotic cells engineered toexpress a recombinant glycoprotein of interest. Specifically, thisdisclosure provides methods for altering the glycosylation patterns of arecombinant glycoprotein of interest by controlling or modulating seeddensity of a cell culture and/or producing the recombinant glycoproteinof interest or culturing eukaryotic cells engineered to express aglycoprotein of interest in a cell culture that has a controlled ormodulated seed density.

In certain embodiments, glycoproteins produced by the methods providedare recovered. The methods are based on the recognition that growth ofcells expressing a recombinant glycoprotein of interest in cell culturemedium with a controlled or modulated seed density can result inalterations to eukaryotic cell glycosylation patterns, such as the levelof afucosylation, galactosylation, N-glycan charge, N-glycolylneuraminicacid (NGNA), and FcγRIIIa binding. In certain embodiments, thealteration of the glycosylation pattern of the recombinant glycoproteinof interest comprises one or more of a reduced level of afucosylation, areduced level of galactosylation, a reduced level ofgalactose-alpha-1,3-galactose (α-gal), a reduced level ofN-glycolylneuraminic acid (NGNA), reduced FcγRIIIa binding, reducedantibody-dependent cell-mediated cytotoxicity, or an increased N-glycancharge. In certain embodiments, the alteration of the glycosylationpattern of the recombinant glycoprotein of interest comprises anincreased level of afucosylation.

As the seed density of the cell culture has differential effects on bothglycosylation and culture conditions, seed density can be used tocontrol or modulate glycosylation patterns and one or more undesirableside effects. In certain embodiments, the control or modulation of cellculture seed density comprises a reduction in the seed density to resultin an increased levels of afucosylation. In certain embodiments, themodulation of cell culture seed density comprises an increase in theseed density to result in a decreased levels of afucosylation.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bysupplementing a culture medium in which the cells are growing with anadditive and controlling or modulating the seed density of the cellculture. In certain embodiments, the disclosure provides methods forproducing the recombinant glycoprotein of interest with an additive in aculture medium that has controlled or modulated cell culture seeddensity, or culturing eukaryotic cells engineered to express aglycoprotein of interest in a tissue culture medium that has beensupplemented with an additive and has a controlled or modulated seeddensity. For example, in certain embodiments a desirable glycosylationpattern is achieved (e.g., a reduced percentage of afucosylatedN-glycans) without substantially increasing the levels of α-gal, whichin some instances can be an undesirable side effect. In otherembodiments, desirable glycosylation patterns are achieved withoutsubstantially reducing sialic acid levels, which can be an undesirableside effect. The desirability of each combination depends on theapplication. If a product quality attribute is prioritized over theproduct quality attribute affected by the corresponding side effect,then providing the additive would still be desirable.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bysupplementing the medium in which the cells are growing withmycophenolic acid and/or mycophenolic acid acyl glucuronide as anadditive and modifying the cell culture seed density. In certainembodiments, the disclosure provides methods for producing therecombinant glycoprotein of interest with mycophenolic acid and/ormycophenolic acid acyl glucuronide as an additive in a culture mediumthat has controlled or manipulated cell culture seed density, orculturing eukaryotic cells engineered to express a glycoprotein ofinterest in a tissue culture medium that has been supplemented withmycophenolic acid and/or mycophenolic acid acyl glucuronide as anadditive and has a controlled or modulated seed density.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bysupplementing the medium in which the cells are growing with insulin asan additive and modifying the cell culture seed density. In certainembodiments, the disclosure provides methods for producing therecombinant glycoprotein of interest with insulin as an additive in aculture medium that has controlled or manipulated cell culture seeddensity, or culturing eukaryotic cells engineered to express aglycoprotein of interest in a tissue culture medium that has beensupplemented with insulin as an additive and has a controlled ormodulated seed density.

In certain embodiments, the disclosure provides methods for altering theglycosylation patterns of a recombinant glycoprotein of interest bymodifying the cell culture seed density and temperature. In certainembodiments, the disclosure provides methods for producing therecombinant glycoprotein of interest in a culture medium that hascontrolled or manipulated cell culture seed density and temperature, orculturing eukaryotic cells engineered to express a glycoprotein ofinterest in a tissue culture medium that has a controlled or modulatedcell culture seed density and temperature. In other words, the variouspossible additive components can be used as levers to achieve the mostdesirable balance between approaching a particular glycosylation patternand minimizing side effects.

The present invention is also applicable to altering, manipulating, orcontrolling the glycosylation pattern of a recombinant glycoprotein ofinterest to match, substantially match, approach, or more closelyresemble the glycosylation pattern of the same glycoprotein, butproduced in a different cell culture system. Recombinant glycoproteinsof interest can be produced according to the invention using variousdifferent cell culture systems, e.g., a batch culture, fed-batch culturea perfusion culture, a shake flask, and/or a bioreactor. In oneembodiment, cells expressing a recombinant glycoprotein of interest arecultured in basal medium to which the additive is introduced as a bolus,or two or more boli, from a stock solution. In another embodiment, theadditive is introduced as a component of a feed medium.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

In certain embodiments, the controlling or modulating of cell cultureseed density comprises having the culture seed density less than regularseed density (“low seed density”). In certain embodiments, thecontrolling or modulating of cell culture seed density comprises havingthe culture seed density to more than regular seed density (“high seeddensity”). For example, the cell culture seed density comprises regularseed density of 3.5-5.5e5 vc/mL for DUXB11 cell line; the cell cultureseed density comprises low seed density of <3e5 vc/mL for DUXB11 cellline; and the cell culture seed density comprises high seed densityof >7e5 vc/mL for DUXB11 cell line. A person of ordinary skill in theart can determine a high seed density, a low seed density and a regularseed density for other cell lines.

The present invention further provides a cell culture compositioncomprising a medium described herein and cells, produced by the methodsprovided herein.

In one embodiment, a cell culture composition produced by the providedmethods can be a batch culture, fed-batch culture or a perfusionculture. In a specific embodiment, a cell culture composition of theinvention is a fed batch culture.

In one embodiment, a cell culture composition produced by the providedmethods comprises mammalian cells selected from the group consisting ofCHO cells, HEK cells, NSO cells, PER.C6 cells, 293 cells, HeLa cells,and MDCK cells. In a specific embodiment, a cell culture compositiondescribed herein comprises CHO cells. In another specific embodiment, acell culture composition described herein comprises HEK cells. Inanother specific embodiment, a cell culture composition described hereincomprises hybridoma cells.

A cell culture composition produced by the provided methods can comprisecells that have been adapted to grow in serum free medium, animalprotein free medium or chemically defined medium. Or it can comprisecells that have been genetically modified to increase their life-span inculture. In one embodiment, the cells have been modified to express ananti-apoptotic gene. In a specific embodiment, the cells have beenmodified to express the bcl-xL antiapoptotic gene. Additionalanti-apoptotic genes that can be used in accordance with the presentinvention include, but are not limited to, E1B-9K, Aven, Mcl.

The present invention provides a method of culturing cells, comprisingcontacting the cells with a medium disclosed herein, supplementing themedium as described above, or culturing cells in a medium supplementedas described above.

Cell cultures can be cultured in a batch culture, fed batch culture or aperfusion culture. In one embodiment, a cell culture according to amethod of the present invention is a batch culture. In anotherembodiment, a cell culture according to a method of the presentinvention is a fed batch culture. In a further embodiment, a cellculture according to a method of the present invention is a perfusionculture. In certain embodiments the cell culture is maintained in ashake flask, in certain embodiments the cell culture is maintained in abioreactor.

In one embodiment, a cell culture according to a method of the presentinvention is a serum-free culture. In another embodiment, a cell cultureaccording to a method of the present invention is a chemically definedculture. In a further embodiment, a cell culture according to a methodof the present invention is an animal protein free culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a medium described herein during the growth phase of theculture. In another embodiment, a cell culture is contacted with amedium described herein during the production phase of the culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a feed medium described herein during the productionphase of the culture. In one embodiment, the culture is supplementedwith the feed medium between about 1 and about 25 times during thesecond time period. In another embodiment, a culture is supplementedwith the feed medium between about 1 and about 20 times, between about 1and about 15 times, or between about 1 and about 10 times during thefirst time period. In a further embodiment, a culture is supplementedwith the feed medium at least once, at least twice, at least threetimes, at least four times, at least five times, at least 6 times, atleast 7 times, at least 8 times, at least 9 times, at least 10 times, atleast 1 times, at least 12 times, at least 13 times, at least 14 times,at least 15 times, at least 20 times, at least 25 times. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein at regular intervals. In one embodiment, theregular interval is about once a day, about once every two days, aboutonce every three days, about once every 4 days, or about once every 5days. In a specific embodiment, the culture is a fed batch culture. Inanother specific embodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein on an as needed basis based on the metabolicstatus of the culture. In one embodiment, a metabolic marker of a fedbatch culture is measured prior to supplementing the culture with a feedmedium described herein. In one embodiment, the metabolic marker isselected from the group consisting of: lactate concentration, ammoniumconcentration, alanine concentration, glutamine concentration, glutamateconcentration, cell specific lactate production rate to the cellspecific glucose uptake rate ratio (LPR/GUR ratio), and Rhodamine 123specific cell fluorescence. In one embodiment, an LPR/GUR value of >0.1indicates the need to supplement the culture with a feed mediumdescribed herein. In a further specific embodiment, a lactateconcentration of >3 g/L indicates the need to supplement the culturewith a feed medium described herein. In another embodiment, a cultureaccording to the present invention is supplemented with a feed mediumdescribed herein when the LPR/GUR value of the culture is >0.1 or whenthe lactate concentration of the culture is >3 g/L. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

In one embodiment, a medium described herein is a feed medium for a fedbatch cell culture. A skilled artisan understands that a fed batch cellculture can be contacted with a feed medium more than once. In oneembodiment, a fed batch cell culture is contacted with a mediumdescribed herein only once. In another embodiment, a fed batch cellculture is contacted with a medium described herein more than once, forexample, at least twice, at least three times, at least four times, atleast five times, at least six times, at least seven times, or at leastten times.

In accordance with the present invention, the total volume of feedmedium added to a cell culture should optimally be kept to a minimalamount. For example, the total volume of the feed medium added to thecell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45 or 50% of the volume of the cell culture prior to adding the feedmedium.

Cell cultures produced by the provided methods can be grown to achieve aparticular cell density, depending on the needs of the practitioner andthe requirement of the cells themselves, prior to being contacted with amedium described herein. In one embodiment, the cell culture iscontacted with a medium described herein at a viable cell density of 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99 percent of maximal viable cell density. In a specificembodiment, the medium is a feed medium.

Cell cultures produced by the provided methods can be allowed to growfor a defined period of time before they are contacted with a mediumdescribed herein. In one embodiment, the cell culture is contacted witha medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 of the cell culture. In another embodiment, the cell culture iscontacted with a medium described herein at week 1, 2, 3, 4, 5, 6, 7, or8 of the cell culture. In a specific embodiment, the medium is a feedmedium.

Cell cultures produced by the provided methods can be cultured in theproduction phase for a defined period of time. In one embodiment, thecell culture is contacted with a feed medium described herein at day 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the production phase.

A culture produced by the provided methods can be maintained inproduction phase for between about 1 day and about 30 days. In oneembodiment, a culture is maintained in production phase for betweenabout 1 day and about 30 days, between about 1 day and about 25 days,between about 1 day and about 20 days, about 1 day and about 15 days,about 1 day and about 14 days, about 1 day and about 13 days, about 1day and about 12 days, about 1 day and about 11 days, about 1 day andabout 10 days, about 1 day and about 9 days, about 1 day and about 8days, about 1 day and about 7 days, about 1 day and about 6 days, about1 day and about 5 days, about 1 day and about 4 days, about 1 day andabout 3 days, about 2 days and about 25 days, about 3 days and about 25days, about 4 days and about 25 days, about 5 days and about 25 days,about 6 days and about 25 days, about 7 days and about 25 days, about 8days and about 25 days, about 9 days and about 25 days, about 10 daysand about 25 days, about 15 days and about 25 days, about 20 days andabout 25 days, about 2 days and about 30 days, about 3 days and about 30days, about 4 days and about 30 days, about 5 days and about 30 days,about 6 days and about 30 days, about 7 days and about 30 days, about 8days and about 30 days, about 9 days and about 30 days, about 10 daysand about 30 days, about 15 days and about 30 days, about 20 days andabout 30 days, or about 25 days and about 30 days. In anotherembodiment, a culture is maintained in production phase for at leastabout 1 day, at least about 2 days, at least about 3 days, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days, at least about 12 days, at leastabout 15 days, at least about 20 days, at least about 25 days, or atleast about 30 days. In a further embodiment, a culture is maintained inproduction phase for about 1 day, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 15 days, about20 days, about 25 days, or about 30 days.

The present invention further provides a method of producing arecombinant glycoprotein interest, comprising culturing cells engineeredto express the recombinant glycoprotein of interest in a culturecomprising a medium described herein; and recovering or isolating therecombinant glycoprotein of interest from the culture. In certainembodiments, the recombinant glycoprotein of interest is an enzyme,receptor, antibody, immunoadhesin, hormone, regulatory factor, antigen,coagulation factor, or binding agent. In a specific embodiment, therecombinant glycoprotein of interest is an antibody. In anotherembodiment, the recombinant glycoprotein of interest is animmunoadhesin. In another embodiment, the recombinant glycoprotein ofinterest is a coagulation factor.

In a specific embodiment, a method of producing a recombinantglycoprotein of interest according to the present invention produces amaximum glycoprotein titer of at least about 0.05 g/L, at least about0.1 g/L, at least about 0.25 g/L, at least about 0.5 g/L, at least about0.75 g/L, at least about 1.0 g/L, at least about 1.5 g/L, at least about2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, atleast about 3.5 g/liter, at least about 4 g/liter, at least about 4.5g/liter, at least about 5 g/liter, at least about 6 g/liter, at leastabout 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, orat least about 10 g/liter. In another embodiment, the method accordingto the present invention produces a maximum glycoprotein titer ofbetween about 1 g/liter and about 10 g/liter, about 1.5 g/liter andabout 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In a specificembodiment, the glycoprotein is an antibody. In another embodiment, theglycoprotein is a blood clotting factor.

The invention further provides a conditioned cell culture mediumproduced by a method described herein.

In one embodiment, a conditioned cell culture medium produced accordingto the provided methods comprises a recombinant glycoprotein ofinterest. In a specific embodiment, a conditioned cell culture mediumaccording to the invention comprises a recombinant glycoprotein ofinterest at a titer of at least about 2 g/liter, at least about 2.5g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at leastabout 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter,at least about 6 g/liter, at least about 7 g/liter, at least about 8g/liter, at least about 9 g/liter, or at least about 10 g/liter, or atiter of between about 1 g/liter and about 10 g/liter, about 1.5 g/literand about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In anotherembodiment, a conditioned cell culture medium according to the inventioncomprises a recombinant glycoprotein at a higher titer than the titerobtained without the use of a medium described herein. In a specificembodiment, the protein or polypeptide is an antibody.

Glycoproteins

Any glycoprotein that is expressible in a host cell can be produced inaccordance with the present invention. The glycoprotein can be expressedfrom a gene that is endogenous to the host cell, or from a gene that isintroduced into the host cell through genetic engineering. Theglycoprotein can be one that occurs in nature, or can alternatively havea sequence that was engineered or selected by the hand of man. Anengineered glycoprotein can be assembled from other glycoproteinsegments that individually occur in nature, or can include one or moresegments that are not naturally occurring.

Glycoproteins that can desirably be expressed by the methods providedherein will often be selected on the basis of an interesting biologicalor chemical activity. For example, the present invention can be employedto express any pharmaceutically or commercially relevant enzyme,receptor, antibody, immunoadhesin, hormone, regulatory factor, antigen,binding agent, etc.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any antibody that can be expressed in a hostcell can be used in accordance with the present invention. In oneembodiment, the antibody to be expressed is a monoclonal antibody.

Particular antibodies can be made, for example, by preparing andexpressing synthetic genes that encode the recited amino acid sequencesor by mutating human germline genes to provide a gene that encodes therecited amino acid sequences. Moreover, these antibodies can beproduced, e.g., using one or more of the following methods.

Numerous methods are available for obtaining antibodies, particularlyhuman antibodies. One exemplary method includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Thedisplay of Fab's on phage is described, e.g., in U.S. Pat. Nos.5,658,727; 5,667,988; and 5,885,793.

In addition to the use of display libraries, other methods can be usedto obtain an antibody. For example, a protein or a peptide thereof canbe used as an antigen in a non-human animal, e.g., a rodent, e.g., amouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies derived from the genes with the desiredspecificity can be produced and selected. See, e.g., XENOMOUSE™, Greenet al. (1994) Nature Genetics 7:13-21, U.S. 2003-0070185, WO 96/34096,and WO 96/33735.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized or deimmunized.Winter describes an exemplary CDR-grafting method that can be used toprepare humanized antibodies described herein (U.S. Pat. No. 5,225,539).All or some of the CDRs of a particular human antibody can be replacedwith at least a portion of a non-human antibody. In one embodiment, itis only necessary to replace the CDRs required for binding or bindingdeterminants of such CDRs to arrive at a useful humanized antibody thatbinds to an antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L. (1985)Science 229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and byU.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and6,407,213. Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulin Fvvariable regions from at least one of a heavy or light chain. Sources ofsuch nucleic acid are well known to those skilled in the art and, forexample, can be obtained from a hybridoma producing an antibody againsta predetermined target, as described above, from germline immunoglobulingenes, or from synthetic constructs. The recombinant DNA encoding thehumanized antibody can then be cloned into an appropriate expressionvector. In one embodiment, the expression vector comprises apolynucleotide encoding a glutamine synthetase polypeptide. (See, e.g.,Porter et al., Biotechnol Prog 26(5):1446-54 (2010).)

The antibody can include a human Fc region, e.g., a wild-type Fc regionor an Fc region that includes one or more alterations. In oneembodiment, the constant region is altered, e.g., mutated, to modify theproperties of the antibody (e.g., to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, or complement function). For example,the human IgG1 constant region can be mutated at one or more residues,e.g., one or more of residues 234 and 237. Antibodies can have mutationsin the CH2 region of the heavy chain that reduce or alter effectorfunction, e.g., Fc receptor binding and complement activation. Forexample, antibodies can have mutations such as those described in U.S.Pat. Nos. 5,624,821 and 5,648,260. Antibodies can also have mutationsthat stabilize the disulfide bond between the two heavy chains of animmunoglobulin, such as mutations in the hinge region of IgG4, asdisclosed in the art (e.g., Angal et al. (1993) Mol. Immunol.30:105-08). See also, e.g., U.S. 2005-0037000.

In other embodiments, the antibody can be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used in this context, “altered” means havingone or more carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original antibody. Addition ofglycosylation sites to the presently disclosed antibodies can beaccomplished by altering the amino acid sequence to containglycosylation site consensus sequences; such techniques are well knownin the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. These methods aredescribed in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit.Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties presenton the antibodies can be accomplished chemically or enzymatically asdescribed in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys.259:52; Edge et al. (1981) Anal. Biochem. 118:131; and Thotakura et al.(1987) Meth. Enzymol. 138:350). See, e.g., U.S. Pat. No. 5,869,046 for amodification that increases in vivo half-life by providing a salvagereceptor binding epitope.

The antibodies can be in the form of full length antibodies, or in theform of fragments of antibodies, e.g., Fab, F(ab′)₂, Fd, dAb, and scFvfragments. Additional forms include a protein that includes a singlevariable domain, e.g., a camel or camelized domain. See, e.g., U.S.2005-0079574 and Davies et al. (1996) Protein Eng. 9(6):531-7.

In one embodiment, the antibody is an antigen-binding fragment of a fulllength antibody, e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment.Typically, the antibody is a full length antibody. The antibody can be amonoclonal antibody or a mono-specific antibody.

In another embodiment, the antibody can be a human, humanized,CDR-grafted, chimeric, mutated, affinity matured, deimmunized, syntheticor otherwise in vitro-generated antibody, and combinations thereof.

The heavy and light chains of the antibody can be substantiallyfull-length. The protein can include at least one, and preferably two,complete heavy chains, and at least one, and preferably two, completelight chains) or can include an antigen-binding fragment (e.g., a Fab,F(ab′)2, Fv or a single chain Fv fragment). In yet other embodiments,the antibody has a heavy chain constant region chosen from, e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosenfrom, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g.,human IgG1). Typically, the heavy chain constant region is human or amodified form of a human constant region. In another embodiment, theantibody has a light chain constant region chosen from, e.g., kappa orlambda, particularly, kappa (e.g., human kappa).

Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes receptors. Receptorsare typically trans-membrane glycoproteins that function by recognizingan extra-cellular signaling ligand. Receptors typically have a proteinkinase domain in addition to the ligand recognizing domain, whichinitiates a signaling pathway by phosphorylating target intracellularmolecules upon binding the ligand, leading to developmental or metabolicchanges within the cell. In one embodiment, the receptors of interestare modified so as to remove the transmembrane and/or intracellulardomain(s), in place of which there can optionally be attached anIg-domain. In one embodiment, receptors to be produced in accordancewith the present invention are receptor tyrosine kinases (RTKs). The RTKfamily includes receptors that are crucial for a variety of functionsnumerous cell types (see, e.g., Yarden and Ullrich, Ann. Rev. Biochem.57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254, 1990,incorporated herein by reference). Non-limiting examples of RTKs includemembers of the fibroblast growth factor (FGF) receptor family, membersof the epidermal growth factor receptor (EGF) family, platelet derivedgrowth factor (PDGF) receptor, tyrosine kinase with immunoglobulin andEGF homology domains-1 (TIE-1) and TIE-2 receptors (Sato et al., Nature376(6535):70-74 (1995), incorporated herein by reference) and c-Metreceptor, some of which have been suggested to promote angiogenesis,directly or indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895-898,1995). Other non-limiting examples of RTK's include fetal liver kinase 1(FLK-1) (sometimes referred to as kinase insert domain-containingreceptor (KDR) (Terman et al., Oncogene 6:1677-83, 1991) or vascularendothelial cell growth factor receptor 2 (VEGFR-2)), fins-like tyrosinekinase-1 (Flt-1) (DeVries et al. Science 255; 989-991, 1992; Shibuya etal., Oncogene 5:519-524, 1990), sometimes referred to as vascularendothelial cell growth factor receptor 1 (VEGFR-1), neuropilin-1,endoglin, endosialin, and Ax1. Those of ordinary skill in the art willbe aware of other receptors that can be expressed in accordance with thepresent invention.

Growth Factors and Other Signaling Molecules

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. Growth factors are typically glycoproteinsthat are secreted by cells and bind to and activate receptors on othercells, initiating a metabolic or developmental change in the receptorcell.

Non-limiting examples of mammalian growth factors and other signalingmolecules include cytokines; epidermal growth factor (EGF);platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs)such as aFGF and bFGF; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3,TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I and -II (IGF-Iand IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-10; tumornecrosis factor (TNF) alpha and beta; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin, hemopoietic growth factor;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha);mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; neurotrophic factorssuch as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5,or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such asNGF-beta. One of ordinary skill in the art will be aware of other growthfactors or signaling molecules that can be expressed in accordance withthe present invention.

G-Protein Coupled Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. G-protein coupled receptors (GPCRs) areproteins that have seven transmembrane domains. Upon binding of a ligandto a GPCR, a signal is transduced within the cell which results in achange in a biological or physiological property of the cell.

GPCRs, along with G-proteins and effectors (intracellular enzymes andchannels which are modulated by G-proteins), are the components of amodular signaling system that connects the state of intracellular secondmessengers to extracellular inputs. These genes and gene-products arepotential causative agents of disease.

The GPCR protein superfamily now contains over 250 types of paralogues,receptors that represent variants generated by gene duplications (orother processes), as opposed to orthologues, the same receptor fromdifferent species. The superfamily can be broken down into fivefamilies: Family I, receptors typified by rhodopsin and thebeta2-adrenergic receptor and currently represented by over 200 uniquemembers; Family II, the recently characterized parathyroidhormone/calcitonin/secretin receptor family; Family III, themetabotropic glutamate receptor family in mammals; Family IV, the cAMPreceptor family, important in the chemotaxis and development of D.discoideum; and Family V, the fungal mating pheromone receptors such asSTE2.

Cells

Any eukaryotic cell or cell type susceptible to cell culture can beutilized in accordance with the present invention. For example, plantcells, yeast cells, animal cells, insect cells, avian cells or mammaliancells can be utilized in accordance with the present invention. In oneembodiment, the eukaryotic cells are capable of expressing a recombinantprotein or are capable of producing a recombinant or reassortant virus.

Non-limiting examples of mammalian cells that can be used in accordancewith the present invention include BALB/c mouse myeloma line (NSO/1,ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, TheNetherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells ±DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). In one embodiment, the present invention is used in theculturing of and expression of polypeptides from CHO cell lines. In aspecific embodiment, the CHO cell line is the DG44 CHO cell line. In aspecific embodiment, the CHO cell line is the DUXB11 CHO cell line. In aspecific embodiment, the CHO cell line comprises a vector comprising apolynucleotide encoding a glutamine synthetase polypeptide. In a furtherspecific embodiment, the CHO cell line expresses an exogenous glutaminesynthetase gene. (See, e.g., Porter et al., Biotechnol Prog26(5):1446-54 (2010).)

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins can beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

The eukaryotic cells according to the present invention can be selectedor engineered to produce high levels of protein or polypeptide, or toproduce large quantities of virus. Often, cells are geneticallyengineered to produce high levels of protein, for example byintroduction of a gene encoding the recombinant glycoprotein of interestand/or by introduction of control elements that regulate expression ofthe gene (whether endogenous or introduced) encoding the recombinantglycoprotein of interest.

The eukaryotic cells can also be selected or engineered to survive inculture for extended periods of time. For example, the cells can begenetically engineered to express a polypeptide or polypeptides thatconfer extended survival on the cells. In one embodiment, the eukaryoticcells comprise a transgene encoding the Bcl-2 polypeptide or a variantthereof. See, e.g., U.S. Pat. No. 7,785,880. In a specific embodiment,the cells comprise a polynucleotide encoding the bcl-xL polypeptide.See, e.g., Chiang G G, Sisk W P. 2005. Biotechnology and Bioengineering91(7): 779-792.

The eukaryotic cells can also be selected or engineered to modify itsposttranslational modification pathways. In one embodiment, the cellsare selected or engineered to modify a protein glycolsylation pathway.In a specific embodiment, the cells are selected or engineered toexpress an aglycosylated protein, e.g., an aglycosylated recombinantantibody. In another specific embodiment, the cells are selected orengineered to express an afucosylated protein, e.g., an afucosylatedrecombinant antibody.

The eukaryotic cells can also be selected or engineered to allowculturing in serum free medium.

Media

The cell culture of the present invention is prepared in any mediumsuitable for the particular cell being cultured. In some embodiments,the medium contains e.g., inorganic salts, carbohydrates (e.g., sugarssuch as glucose, galactose, maltose or fructose), amino acids, vitamins(e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin,thiamine and biotin), fatty acids and lipids (e.g., cholesterol andsteroids), proteins and peptides (e.g., albumin, transferrin,fibronectin and fetuin), serum (e.g., compositions comprising albumins,growth factors and growth inhibitors, such as, fetal bovine serum,newborn calf serum and horse serum), trace elements (e.g., zinc, copper,selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzedproteins derived from plant or animal sources), and combinationsthereof. Commercially available media such as 5×-concentrated DMEM/F12(Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed(Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (IrvineScientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMDMillipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed BioreactorSupplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria),ActiCHO (PAA/GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ([DMEM], Sigma) are exemplary nutrient solutions. In addition,any of the media described in Ham and Wallace, (1979) Meth. Enz., 58:44;Barnes and Sato, (1980) Anal. Biochem., 102:255; U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; InternationalPublication Nos. WO 90/03430; and WO 87/00195; the disclosures of all ofwhich are incorporated herein by reference, can be used as culturemedia. Any of these media can be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as gentamycin), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range) lipids (such as linoleic or other fatty acids) andtheir suitable carriers, and glucose or an equivalent energy source. Insome embodiments the nutrient media is serum-free media, a protein-freemedia, or a chemically defined media. Any other necessary supplementscan also be included at appropriate concentrations that would be knownto those skilled in the art.

In one embodiment, the mammalian host cell is a CHO cell and a suitablemedium contains a basal medium component such as a DMEM/HAM F-12 basedformulation (for composition of DMEM and HAM F12 media, see culturemedia formulations in American Type Culture Collection Catalogue of CellLines and Hybridomas, Sixth Edition, 1988, pages 346-349) with modifiedconcentrations of some components such as amino acids, salts, sugar, andvitamins, recombinant human insulin, hydrolyzed peptone, such asPrimatone HS or Primatone RL (Sheffield, England), or the equivalent; acell protective agent, such as Pluronic F68 or the equivalent pluronicpolyol; gentamycin; and trace elements.

The present invention provides a variety of media formulations that,when used in accordance with other culturing steps described herein,minimize or prevent decreases in cellular viability in the culture whileretaining the ability to manipulate, alter, or change glycosylation of arecombinant glycoprotein of interest.

A media formulation of the present invention that has been shown to beto useful in manipulating glycosylation, while not having greatlynegative impacts on metabolic balance, cell growth and/or viability oron expression of polypeptide or protein comprises the media supplementsdescribed herein. One of ordinary skill in the art will understand thatthe media formulations of the present invention encompass both definedand non-defined media.

Cell Culture Processes

Various methods of preparing mammalian cells for production of proteinsor polypeptides by batch and fed-batch culture are well known in theart. A nucleic acid sufficient to achieve expression (typically a vectorcontaining the gene encoding the polypeptide or protein of interest andany operably linked genetic control elements) can be introduced into thehost cell line by any number of well-known techniques. Typically, cellsare screened to determine which of the host cells have actually taken upthe vector and express the polypeptide or protein of interest.Traditional methods of detecting a particular polypeptide or protein ofinterest expressed by mammalian cells include but are not limited toimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linkedimmunosorbentassay (ELISA), high performance liquid chromatography(HPLC) techniques, biological activity assays and affinitychromatography. One of ordinary skill in the art will be aware of otherappropriate techniques for detecting expressed polypeptides or proteins.If multiple host cells express the polypeptide or protein of interest,some or all of the listed techniques can be used to determine which ofthe cells expresses that polypeptide or protein at the highest levels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well-known to one of ordinary skill in the art. The cellexpressing the polypeptide of interest is typically propagated bygrowing it at a temperature and in a medium that is conducive to thesurvival, growth and viability of the cell. The initial culture volumecan be of any size, but is often smaller than the culture volume of theproduction bioreactor used in the final production of the polypeptide orprotein of interest, and frequently cells are passaged several times inbioreactors of increasing volume prior to seeding the productionbioreactor. The cell culture can be agitated or shaken to increaseoxygenation of the medium and dispersion of nutrients to the cells.Alternatively or additionally, special sparging devices that are wellknown in the art can be used to increase and control oxygenation of theculture. In accordance with the present invention, one of ordinary skillin the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor, including butnot limited to pH, temperature, oxygenation, etc.

The cell density useful in the methods of the present invention can bechosen by one of ordinary skill in the art. In accordance with thepresent invention, the cell density can be as low as a single cell perculture volume. In some embodiments of the present invention, startingcell densities (seed density) can range from about 2×10² viable cellsper mL to about 2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 5×10⁶ or 10×10⁶ viable cellsper mL and higher.

In accordance with the present invention, a cell culture size can be anyvolume that is appropriate for production of polypeptides. In oneembodiment, the volume of the cell culture is at least 500 liters. Inother embodiments, the volume of the production cell culture is 10, 50,100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, 12,000 liters or more,or any volume in between. For example, a cell culture will be 10 to5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters,500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000to 10,000 liters, or 1,000 to 15,000 liters. Or a cell culture will bebetween about 500 liters and about 30,000 liters, about 500 liters andabout 20,000 liters, about 500 liters and about 10,000 liters, about 500liters and about 5,000 liters, about 1,000 liters and about 30,000liters, about 2,000 liters and about 30,000 liters, about 3,000 litersand about 30,000 liters, about 5,000 liters and about 30,000 liters, orabout 10,000 liters and about 30,000 liters, or a cell culture will beat least about 500 liters, at least about 1,000 liters, at least about2,000 liters, at least about 3,000 liters, at least about 5,000 liters,at least about 10,000 liters, at least about 15,000 liters, or at leastabout 20,000 liters.

One of ordinary skill in the art will be aware of and will be able tochoose a suitable culture size for use in practicing the presentinvention. The production bioreactor for the culture can be constructedof any material that is conducive to cell growth and viability that doesnot interfere with expression or stability of the produced polypeptideor protein.

The temperature of the cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable. Forexample, during the initial growth phase, CHO cells grow well at 37° C.In general, most mammalian cells grow well within a range of about 25°C. to 42° C.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturecan be steadily increased or decreased during the initial growth phase.Alternatively, the temperature can be increased or decreased by discreteamounts at various times during the initial growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperatures should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells can be grown during the initial growth phase for a greater orlesser amount of time, depending on the needs of the practitioner andthe requirement of the cells themselves. In one embodiment, the cellsare grown for a period of time sufficient to achieve a viable celldensity that is a given percentage of the maximal viable cell densitythat the cells would eventually reach if allowed to grow undisturbed.For example, the cells can be grown for a period of time sufficient toachieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximalviable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are grown, and theintrinsic growth rate of the cells, the cells can be grown for 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moredays. In some cases, the cells can be allowed to grow for a month ormore. In one embodiment, the growth phase is between about 1 day andabout 20 days, about 1 day and about 15 days, about 1 day and about 14days, about 1 day and about 13 days, about 1 day and about 12 days,about 1 day and about 11 days, about 1 day and about 10 days, about 1day and about 9 days, about 1 day and about 8 days, about 1 day andabout 7 days, about 1 day and about 6 days, about 1 day and about 5days, about 1 day and about 4 days, about 1 day and about 3 days, about2 days and about 15 days, about 3 days and about 15 days, about 4 daysand about 15 days, about 5 days and about 15 days, about 6 days andabout 15 days, about 7 days and about 15 days, about 8 days and about 15days, about 9 days and about 15 days, about 10 days and about 15 days,about 2 days and about 20 days, about 3 days and about 20 days, about 4days and about 20 days, about 5 days and about 20 days, about 6 days andabout 20 days, about 7 days and about 20 days, about 8 days and about 20days, about 9 days and about 20 days, about 10 days and about 20 days,or about 10 days and about 20 days. In another embodiment, the growthphase is at least about 1 day, at least about 2 days, at least about 3days, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 15 days, or at least about 20 days. In a furtherembodiment, the growth phase is about 1 day, about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 15days, or about 20 days.

The cells would be grown for 0 days in the production bioreactor iftheir growth in a seed bioreactor, at the initial growth phasetemperature, was sufficient that the viable cell density in theproduction bioreactor at the time of its inoculation is already at thedesired percentage of the maximal viable cell density. The practitionerof the present invention will be able to choose the duration of theinitial growth phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

The cell culture can be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base and oxygenation can be controlledwith sparging devices that are well known in the art.

In one embodiment, at the end of the initial growth phase, at least oneof the culture conditions is shifted so that a second set of cultureconditions is applied. The shift in culture conditions can beaccomplished by a change in the temperature, pH, osmolality or chemicalinductant level of the cell culture. In one embodiment, the cultureconditions are shifted by shifting the temperature of the culture.

When shifting the temperature of the culture, the temperature shift canbe relatively gradual. For example, it can take several hours or days tocomplete the temperature change. Alternatively, the temperature shiftcan be relatively abrupt. For example, the temperature change can becomplete in less than several hours. Given the appropriate productionand control equipment, such as is standard in the commercial large-scaleproduction of polypeptides or proteins, the temperature change can evenbe complete within less than an hour.

The temperature of the cell culture in the subsequent growth phase willbe selected based primarily from the range of temperatures at which thecell culture remains viable and expresses recombinant polypeptides orproteins at commercially adequate levels with the desired level ofglycosylation. In general, most mammalian cells remain viable andexpress recombinant polypeptides or proteins at commercially adequatelevels within a range of about 25° C. to 42° C. In one embodiment,mammalian cells remain viable and express recombinant polypeptides orproteins at commercially adequate levels within a range of about 25° C.to 35° C. Those of ordinary skill in the art will be able to selectappropriate temperature or temperatures in which to grow cells,depending on the needs of the cells and the production requirements ofthe practitioner.

In accordance with the present invention, once the conditions of thecell culture have been shifted as discussed above, the cell culture ismaintained for a subsequent production phase under a second set ofculture conditions conducive to the survival and viability of the cellculture and appropriate for expression of the desired polypeptide orprotein at commercially adequate levels.

As discussed above, the culture can be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In oneembodiment, the temperature of the culture is shifted. According to thisembodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Forexample, during the subsequent production phase, CHO cells expressrecombinant polypeptides and proteins well within a range of 25° C. to35° C.

In accordance with the present invention, the cells can be maintained inthe subsequent production phase until a desired cell density orproduction titer is reached. In one embodiment, the cells are maintainedin the subsequent production phase until the titer to the recombinantpolypeptide or protein reaches a maximum. In other embodiments, theculture can be harvested prior to this point, depending on theproduction requirement of the practitioner or the needs of the cellsthemselves. For example, the cells can be maintained for a period oftime sufficient to achieve a viable cell density of 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percentof maximal viable cell density. In some cases, it is desirable to allowthe viable cell density to reach a maximum, and then allow the viablecell density to decline to some level before harvesting the culture. Inan extreme example, it can be desirable to allow the viable cell densityto approach or reach zero before harvesting the culture.

In another embodiment of the present invention, the cells are allowed togrow for a defined period of time during the subsequent productionphase. For example, depending on the concentration of the cell cultureat the start of the subsequent growth phase, the temperature at whichthe cells are grown, and the intrinsic growth rate of the cells, thecells can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days. In some cases, the cells can beallowed to grow for a month or more. The practitioner of the presentinvention will be able to choose the duration of the subsequentproduction phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

In certain cases, it can be beneficial or necessary to supplement thecell culture during the growth and/or subsequent production phase withnutrients or other medium components that have been depleted ormetabolized by the cells. For example, it might be advantageous tosupplement the cell culture with nutrients or other medium componentsobserved to have been depleted. Alternatively or additionally, it can bebeneficial or necessary to supplement the cell culture prior to thesubsequent production phase. As non-limiting examples, it can bebeneficial or necessary to supplement the cell culture with hormonesand/or other growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source.

These supplementary components, including the amino acids, can all beadded to the cell culture at one time, or they can be provided to thecell culture in a series of additions. In one embodiment of the presentinvention, the supplementary components are provided to the cell cultureat multiple times in proportional amounts. In another embodiment, it canbe desirable to provide only certain of the supplementary componentsinitially, and provide the remaining components at a later time. In yetanother embodiment of the present invention, the cell culture is fedcontinually with these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the medium or solution containing the supplementarycomponents added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell cultureprior to providing the supplementary components.

The cell culture can be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base and oxygenation can becontrolled with sparging devices that are well known in the art.

In certain embodiments of the present invention, the practitioner canfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase.

In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal canpotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it can be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density can be measured using ahemacytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density can be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, Biuret assays, and UV absorbance. It can also be beneficial ornecessary to monitor the posttranslational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

The practitioner can also monitor the metabolic status of the cellculture, for example, by monitoring the glucose, lactate, ammonium, andamino acid concentrations in the cell culture, as well as by monitoringthe oxygen production or carbon dioxide production of the cell culture.For example, cell culture conditions can be analyzed by using NOVABioprofile 100 or 400 (NOVA Biomedical, WA). Additionally, thepractitioner can monitor the metabolic state of the cell culture bymonitoring the activity of mitochondria. In embodiment, mitochondrialactivity can be monitored by monitoring the mitochondrial membranepotential using Rhodamine 123. Johnson L V, Walsh M L, Chen L B. 1980.Proceedings of the National Academy of Sciences 77(2):990-994.

Isolation of Expressed Polypeptide

In general, it will typically be desirable to isolate and/or purifyproteins or polypeptides expressed according to the present invention.In one embodiment, the expressed polypeptide or protein is secreted intothe medium and thus cells and other solids can be removed, as bycentrifugation or filtering for example, as a first step in thepurification process.

Alternatively, the expressed polypeptide can be bound to the surface ofthe host cell. In this embodiment, the media is removed and the hostcells expressing the polypeptide or protein are lysed as a first step inthe purification process. Lysis of mammalian host cells can be achievedby any number of means well known to those of ordinary skill in the art,including physical disruption by glass beads and exposure to high pHconditions.

The polypeptide can be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins (See, e.g., Scopes, Protein Purification Principles andPractice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J.and Hames, B. D. (eds.), Protein Expression: A Practical Approach,Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J.N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methodsin Enzymology Series, Vol 182), Academic Press, 1997, all incorporatedherein by reference). For immunoaffinity chromatography in particular,the protein can be isolated by binding it to an affinity columncomprising antibodies that were raised against that protein and wereaffixed to a stationary support. Alternatively, affinity tags such as aninfluenza coat sequence, poly-histidine, or glutathione-S-transferasecan be attached to the protein by standard recombinant techniques toallow for easy purification by passage over the appropriate affinitycolumn. Protease inhibitors such as phenyl methyl sulfonyl fluoride(PMSF), leupeptin, pepstatin or aprotinin can be added at any or allstages in order to reduce or eliminate degradation of the polypeptide orprotein during the purification process. Protease inhibitors areparticularly desired when cells must be lysed in order to isolate andpurify the expressed polypeptide or protein. One of ordinary skill inthe art will appreciate that the exact purification technique will varydepending on the character of the polypeptide or protein to be purified,the character of the cells from which the polypeptide or protein isexpressed, and the composition of the medium in which the cells weregrown.

Pharmaceutical Compositions

A polypeptide can be formulated as a pharmaceutical composition foradministration to a subject, e.g., to treat or prevent a disorder ordisease. Typically, a pharmaceutical composition includes apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (See e.g., Berge, S. M., etal. (1977) Pharm. Sci. 66:1-19). In one embodiment, a pharmaceuticalcomposition is an immunogenic composition comprising a virus produced inaccordance with methods described herein.

Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington. The Science and Practiceof Pharmacy, 20^(th) ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) Ed., Lippincott Williams & Wilkins Publishers (1999)(ISBN: 0683305727); and Kibbe (ed.), Handbook of PharmaceuticalExcipients American Pharmaceutical Association, 3^(rd) ed. (2000) (ISBN:091733096X).

The pharmaceutical compositions can be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form can depend on the intended mode of administration andtherapeutic application. Typically compositions for the agents describedherein are in the form of injectable or infusible solutions.

In one embodiment, the antibody is formulated with excipient materials,such as sodium chloride, sodium dibasic phosphate heptahydrate, sodiummonobasic phosphate, and a stabilizer. It can be provided, for example,in a buffered solution at a suitable concentration and can be stored at2-8° C.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze drying that yield a powder of an agent describedherein plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the polypeptide can be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known. See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York(1978).

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES

The following materials and methods are provided for the experimentsoutlined below.

Cell line: The cell line used in this study produced a growth factorreceptor immunoadhesin polypeptide. The cell line was constructed usingDG44 adapted to grow in serum-free medium (Prentice, 2007). The cellline DUXB11 was also used in this study.

Cell culture methods: Cells were thawed and maintained as in a previousreport (Kshirsagar, et al. 2012 Biotechnol Bioeng, Huang, et al.Biotechnology Progress 26(5):1400-1410 (2010)). Basal medium for thawand passing was the same as in previous reports (Kshirsagar/Huang).Briefly, cells were thawed and maintained in 3 L shake flasks (CorningLife Sciences, Corning, N.Y.) with 1 L working volumes and were passagedevery 2-3 days. For maintenance cultures the incubator was controlled at37° C. and 5% CO2.

Bioreactor culture conditions: Fed batch cultures were performed in 5 Lglass Applikon vessels using Finesse TruBio DV controllers (FinesseSolutions, San Jose, Calif.) with an initial working volume between2-2.5 L. Bioreactors were seeded at constant seed density of 4×10⁵cells/ml. Concentrated feed medium was delivered on days 3, 5, and everyday or every other day following through harvest. Temperature wasmaintained at 35° C. or 36° C. and pH was controlled at 7.1+/−0.2 by theaddition of either 1 M sodium carbonate or carbon dioxide. Dissolvedoxygen was maintained at 30%-40% by air and oxygen sparge using adrilled hole sparger. Agitation was maintained between 200-400 RPMthroughout the culture to limit total gas flow, while an overlay wasmaintained between 0.005 and 0.04 vvm.

Offline analysis: Samples were taken on most days and analyzed with avariety of equipment. Cell density and viability were measured using thestandard technique of trypan blue exclusion using a Cedex (RocheInnovatis AG, Germany). Cell viability and growth rate of the variouscultures were measured during the culture time course at specific dayspost-inoculation.

Example 1—Effect of Copper (II) Sulfate, Hypoxanthine, Glucosamine, andGalactose on Cell Culture Performance

Copper (II) sulfate, hypoxanthine, guanine, glucosamine, and galactosewere delivered to two different immunoadhesin-expressing CHO cellcultures either as part of the feed media or as boli from distinct stocksolutions. The typical culture process is a 14-day process that consistsof a growth phase from Day 0 to Day 6 at 37° C. and a protein productionphase from Day 6 to Day 14 at 30° C.

Addition of these components to cell culture media negatively affectedthe culture density or viability as compared to the control condition tovarious degrees (FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B,FIG. 4A, FIG. 4B, FIG. 5A, FIG. 5B). The data provided is segregatedbased on the cell line and format/type of culture vessel used (shakeflask versus bioreactor).

Example 2—Effect of Glucosamine and Galactose on the Percentage ofAfucose and NGNA

The effects of glucosamine and galactose were examined using bothCHO-043 cells (Table 1) and CHO-602 (Table 2). In CHO-043 cells,glucosamine alone greatly reduced sialylation from 12 mol/chain to 6mol/chain. It also reduced the level of α-gal. The combination ofglucosamine and galactose generated control-like sialylation levelswhile significantly reducing % NGNA. The combination did not generate asignificant increase in % α-gal compared to control.

TABLE 1 Effect of glucosamine and galactose on CHO-043 % afucose, %α-gal, sialylation, and % NGNA (fed-batch shake flask). N-glycan andsialylation values were obtained from Day 12 samples purified using ProApurification. N-glycans were then enzymatically removed from purifiedprotein and analyzed using HILIC-UPLC. ProG titer values were alsoobtained from Day 12 harvest samples. CHO-043 Fed-batch TSA %α- TiterShake Flask % afucose (mol/chain) % NGNA gal (g/L) Comparator <5.1%  NoData  <1%  <1% No Data polypeptide CHO-043 Control 2.79%  12.3 4.6% 7.2%2.0 20 mM GlcN 1.7%  6.6 3.8% 2.4% 1.8 (D2, D4 Boli) GlcN and 2.1% 12.02.2% 8.1% 1.9 Galactose

In CHO-602, glucosamine alone had little effect on N-glycan parameterssuch as % afucose and % NGNA. It did, however, reduce sialylation. Butwhen used in concert with galactose, it reduced % afucose and % NGNA(Table 2).

TABLE 2 Effect of glucosamine and galactose on CHO-602 % afucose,%α-gal, sialylation, and % NGNA (fed-batch shake flask format). N-glycanand sialylation values were obtained from Day 12 samples purified usingProA purification. N-glycans were then enzymatically removed frompurified protein and analyzed using HILIC-UPLC. ProG titer values wereobtained from Day 14 harvest samples. CHO-602 Fed-batch Shake TSA TiterFlask % afucose (mol/chain) % NGNA (g/L) Comparator <5.1%  No Data  <1%No Data polypeptide CHO-602 Control 9.6 ± 0.3 11.8 ± 0.4 1.6 ± 0.3 1.7 ±0.1 10 mM GlcN 8.9% 11.1 1.3% 1.4 (D6 Bolus) GlcN and Galactose 7.5%11.7 1.1% 1.5

Example 3—Effect of Hypoxanthine and Guanine on FcγRIIIa Binding

Addition of hypoxanthine and guanine to fed-batch shake flask culturesbrought the levels of FcγRIIIa binding within the range of a comparatorpolypeptide produced in a different cell type (Table 3). However, therewas no change in the % afucose levels.

TABLE 3 Effect of hypoxanthine alone on % FcγRIIIa binding (fed-batchshake flask). Day 14 samples were two-column purified using ProA andhydrophobic interaction chromatography (HIC). The reported percentbinding is relative to Enbrel innovator material. ProG titer values werealso obtained from Day 14 harvest samples. Titer Fed-batch Shake Flask %afucose % FcγRIIIa (g/L) Comparator polypeptide <5.1% 85-120% No DataExptA Control 9.8% 125% 1.8 ExptA 1 mM Hypoxanthine (D6 9.5%  85% 1.7Bolus) ExptA 0.2 mM Guanine (D6 Bolus) 10.5%  92% 1.5 ExptB Control 9.6%143% 1.8 ExptB 1 mM Hypoxanthine (D6 8.3% 122% 1.6 Bolus) ExptB 0.2 mMGuanine (D6 Bolus) 10.2% 121% 1.6

Example 4—Effect of Copper (II) Sulfate on N-Linked Glycosylation

Addition of copper (II) sulfate alone moved % afucose, %galactosylation, and % charged levels of N-linked glycosylation towardvalues associated with a comparator polypeptide produced in a differentmammalian cell (Table 4). These data are significant because typically,it is desirable to increase afucose levels as this has a positive effecton bioactive effects such as % FcγRIIIa binding and ADCC.

TABLE 4 Effect of copper (II) sulfate on N-linked glycosylation(fed-batch shake flask). All N- glycan values were obtained from Day 12samples purified using ProA purification. N-glycans were thenenzymatically removed from purified protein and analyzed usingHILIC-UPLC. ProG titer values were obtained from Day 14 harvest samples.Fed-batch % Titer Shake Flask % afucose galactosylation % charged (g/L)Comparator <5.1%  <57.6%  >45.7 No Data polypeptide Control (n = 4) 9.6± 0.3 73.1 ± 2.3 36.7 ± 2.5 1.7 ± 0.1 Feed supp. w/ 1 mM 8.4% 62.7%43.7% 1.8 CuSO₄ 0.2 mM CuSO₄ (D6 8.2% 70.3% 42.1% 1.9 Bolus) 0.5 mMCuSO₄ (D6 8.2% 68.5% 42.4% 1.9 Bolus)

Example 5—Effect of Copper (II) Sulfate, Glucosamine, and Galactose onN-Linked Glycosylation

Addition of copper (II) sulfate alone in the bioreactor confirmed theshake flask results (Table 5). Furthermore, the combination of copper(II) sulfate, glucosamine, galactose favorably reduced % afucose, butcame at the expense of lower % charged levels. Despite the addition ofglucosamine, a known inhibitor of sialylation, sialylation (total sialicacid—TSA) was not significantly lower compared to that of the controlconditions. The addition of galactose likely compensated for theTSA-reducing effect of the glucosamine.

TABLE 5 Effect of copper (II) sulfate, glucosamine, and galactose onN-linked glycosylation (bioreactor). N-glycan values were obtained fromDay 14 samples purified using ProA purification. N-glycans were thenenzymatically removed from purified protein and analyzed usingHILIC-UPLC. ProG titer values were obtained from Day 14 harvest samples.% % % % TSA % Titer 3 L Fed-batch Bioreactor afucose galact. chargedα-gal (mol/chain) NGNA (g/L) Comparator polypeptide  <5.1%  <57.6% >45.7% <1%  No Data  <1% No Data Control (n = 3) 8.1 ± 0.6 71.9 ± 2.435.9 ± 2.7 1.4 ± 0.4 13.3 0.8% 2.0 ± 0.1 Feed w/1 mM CuSO₄ 7.1 65.4 41.90.4 13.5 0.7% 2.0 CuSO4, GlcN, and 5.9 ± 0.8 62.4 ± 2.3 29.0 ± 5.0 0.512.7 ± 0.4 0.8% 2.0 Galactose (n = 2)

Example 6—Effect of Copper (II) Sulfate and Hypoxanthine on N-LinkedGlycosylation

The combination of copper (II) sulfate and hypoxanthine brought aboutthe most favorable PQ results (Table 6). The combination simultaneouslyreduced % afucose, % galactosylation, and % α-gal. CuSO₄ andhypoxanthine generated an N-glycan profile that was similar to thatgenerated by CuSO₄, glucosamine, and galactose (Table 5), but did notgenerate an undesired increase in % α-gal. However, the combination hada deleterious effect on cell growth, viability, and resultant titer.

TABLE 6 Effect of copper (II) sulfate and hypoxanthine on N-linkedglycosylation (bioreactor). N-glycan values were obtained from Day 14samples purified using ProA purification. N- glycans were thenenzymatically removed from purified protein and analyzed usingHILIC-UPLC. ProG titer values were obtained from Day 14 harvest samples.3L Fed-batch %α- Titer Bioreactor % afucose % galact. % charged gal(g/L) Comparator <5.1%  <57.6%  >45.7%   <1% No Data polypeptide Control8.1 ± 0.6 71.9 ± 2.4 35.9 ± 2.7 1.4 ± 0.4 2.0 ± 0.1 (n = 3) CuSO₄ and6.2% 58.9% 40.7% 0.6% 1.4 Hypoxan- thine

TABLE 7 Effect of copper (II) sulfate and Hypoxanthine-Thymidinesupplement on CHO-602 N-linked glycosylation (fed-batch shake flask).Culture conditions were describe in FIG. 6A and FIG. 6B. N-glycan valueswere obtained from Day 14 samples purified using ProA purification.N-glycans were then enzymatically removed from purified protein andanalyzed using HILIC-UPLC. ProG titer values were obtained from Day 14harvest samples. CHO-602 Fed-batch Titer SF % afucose % galact. %charged (g/L) Comparator polypeptide <5.1% <57.6% >45.7%  No DataControl 9.9% 72.4% 26.8% 2.0 g/L CuSO₄ and HT Low 8.4% 59.5% 32.7% 1.9g/L CuSO₄ and HT High 8.9% 49.7%   31% 1.7 g/L

Example 7—Manipulation of Glycosylation

The desirability of each supplementation combination depends on theapplication. If a product quality attribute is prioritized over theproduct quality attribute affected by the corresponding side effect,then adding the supplement would still be desirable.

For instance, if reducing afucosylation is a priority and a drop intiter is acceptable, then copper+hypoxanthine becomes desirable as thatis one of the combinations that generates a dramatic effect onafucosylation. However, if titer is of greatest importance, then themore desirable solution becomes the copper sulfate itself.

In the overall context of minimizing undesired side effects, the orderof desirability is as follows:

1. Copper sulfate

2. Copper sulfate+hypoxanthine+thymidine

3. Copper sulfate+hypoxanthine

4. Copper sulfate+glucosamine+galactose

5. Hypoxanthine

6. Glucosamine+galactose

7. Guanine

8. Glucosamine

Reduce % Afucosylation

Supplement Desirability Side Effect Data GlcN + Galactose Low Unknownsialylated Table 1, 2 species at HILIC elution times of RT29 and RT34Copper + GlcN + Low Unknown sialylated Table 5 Galactose species atHILIC elution times of RT29 and RT34 Copper High None Table 4, 5Copper + Medium Lower titer Table 6 Hypoxanthine Copper + Med-HighSlightly lower titer Table 7 Hypoxanthine + ThymidineReduce % Galactosylation

Supplement Desirability Side Effect Data Copper High None Table 4, 5Copper + GlcN + Low Unknown peaks Table 5 Galactose Copper +Hypoxanthine Medium Lower titer Table 6 Copper + Med-High Slightly lowertiter Table 7 Hypoxanthine + ThymidineIncrease % Charged

Supplement Desirability Side Effect Data Copper High None Table 4, 5Copper + Hypoxanthine Medium Lower titer Table 6 Copper + Med-HighSlightly lower titer Table 7 Hypoxanthine + ThymidineReduce % α-Gal

Supplement Desirability Side Effect Data GlcN Low Reduce TSA Table 1

Reduce NGNA

Supplement Desirability Side Effect Data GlcN + Galactose High NoneTable 1

Example 8—Effect of Mycophenolic Acid (MPA) on Afucosylation (Desired),and Cell Titer and Cell Viability (Side Effect) in Shake Flask

DUXB11 cell lines (cell line #1 and #2) were cultured in 3 L bioreactorrespectively for 7 days following the platform process. On day 7, thecells were divided into several 1 L shake flasks with 200 mL workingvolume and then dosed with various amounts of MPA (0 μM, 10 μM, 30 μM).After dosing MPA, shake flask fed-batch is conducted until day-3harvest. Filtered supernatant samples were analyzed for titer andN-glycan analysis for afucosylation level. The data showed that anincrease in MPA dosing concentration caused an increase in theafucosylation level (desired) (see Table 8 below). However, the titerwas decreased (side effect) (see Table 8 below and FIG. 7A and FIG. 7B).The impact on viability was not significant because this is a quickscreening study happened in shake flask and cells only got 3 days toexpose to MPA. The impact on viability became much more significant inbioreactor process (see Example 9 below).

TABLE 8 Effect of mycophenolic acid supplement on DUXB11 afucosylation(shake flask). Total MPA added Afucosylation (%) Titer (g/L) Cellviability (%)  0 μM 2.25 0.15 63.1  1 μM 3.85 0.136 62 10 μM 4.9 0.10661 30 μM 4.4 0.105 62

Example 9—Effect of Mycophenolic Acid (MPA) Afucosylation (Desired) andCell Titer and Cell Viability (Side Effect) in a Bioreactor

DUXB11 cell lines were cultured in 3 L bioreactor respectively followingthe platform process. On day 6, various amounts of MPA were dosed (0 μMand 5 μM). After dosing MPA, bioreactors were kept running until theywere harvested on day 13. Filtered supernatant samples were analyzed fortiter and N-glycan analysis for afucosylation level. The data showedthat MPA dosing addition increased afucosylation level (desired), butdecreased the cell viability and the titer (side effect), which is thesame trend as that in shake flask study.

TABLE 9 Effect of mycophenolic acid supplement on DUXB11 afucosylation(bioreactor). Total MPA added Afucosylation (%) Titer (g/L) Cellviability (%) 0 μM 2.7 0.24 42 5 μM 8.3 0.28 25

Example 10—Effect of the Timing of Supplementation with MycophenolicAcid (MPA)

The culture conditions are described in FIG. 8. The data showed that theaddition of MPA on the day of peak variable cell density (VCD) had asignificant impact on afucosylation, while the addition of MPA on thedifferent day of peak VCD had non-significant impact on afucosylation.In addition, afucosylation level was increased with peak VCD day MPAaddition (fixed 5 μM MPA vs. VCD based MPA addition). Data indicatedthat MPA dosing time is much more important than dosing amount.

Example 11—Effect of Seed Density on Cell Growth and Afucosylation

The culture conditions are described in FIG. 9A, FIG. 9B, and FIG. 10.The data showed different cell performances due to different seeddensity (FIG. 9A and FIG. 9B). The data also showed that the seeddensity could change the MPA impact on afucosylation level even thoughthe same MPA amount was added (FIG. 10). Normally, the seed density andafucosylation are in inverse proportion. The similar study was carriedout using another DUXB11 cell line, the same trend was observed.

Example 12—Effect of Insulin on Afucosylation and Titer

The culture conditions are described in FIG. 11. The data showed thatincreasing insulin concentration, with same MPA supplementation, led toincreased afucosylation in fed-batch culture (Table 10A and 10B below,and FIG. 11).

TABLE 10A Effect of insulin and MPA supplement on DUXB11 afucosylation.Total insulin added Afucosylation (%) Titer (g/L) 11 mg/L 4.9 2.3 13mg/L 6.2 2.6 17 mg/L 8.1 2.8 24 mg/L 7.8 3.1

TABLE 10B Glycosylation map after insulin and MPA supplement on DUXB11.G1 + G1F + FcgRIII a M3F G0 G0F MS G1′ G1F′ M6 G2 G2F Afucosyl bindingMin 0.4 3.4 37.1 0.8 1.1 26.0 0.0 2.4 5.0 79 Max 4.3 5.5 54.1 3.8 4.042.9 0.9 7.8 9.6 136 2.5 mg/L 1.0 3.9 68.1 2.2 0.9 20.2 0.90 0.0 1.8 4.960 ins in feed 1 10 mg/L 0.9 5.1 64.9 2.2 1.1 21.7 0.9 0.0 2.3 6.2 71ins in feed 1 30 mg/L 0.9 6.7 62.5 2.4 1.3 21.1 1.1 0.0 2.3 8.1 100 insin feed 1 60 mg/L 0.8 6.4 61.4 2.5 1.4 22.9 0.9 0.0 2.6 7.8 100 ins infeed 1

Example 13—Effect of Temperature on Afucosylation (Desired) and Titer,Viability (Side Effects)

The effect of temperature shift and supplementation with MPA is providedin FIG. 12, FIG. 13, FIG. 14A, and FIG. 14B and Table 11 below.

TABLE 11 Effect of temperature and MPA supplementation (Day 5) on DUXB11afucosylation. Temperature (° C.) Afucosylation (%) Titer (g/L) Cellviability (%) 35 to 30 11.9 1.5 43 35 to 33 10.7 1.8 29

Example 14—Effect of Seed Density on Cell Growth and Afucosylation

The effect supplementation with MPA on cell growth, cell viability,viable cell density, and titer is provided in FIG. 15A, FIG. 15B, FIG.15C, FIG. 16A, FIG. 16B, FIG. 16C, FIG. 17A, FIG. 17B, and FIG. 17C.

Example 15—Effect of Mycophenolic Acid (MPA) and Mycophenolic Acid AcylGlucuronide (acMPAG) on Afucosylation in Shake Flask

BIIB603 cell line was scaled up to 5 L BR for fed-batch productionprocess. On day 5, temperature was shifted from 37° C. to 31° C. On day6, cell culture was drained from the bioreactor and divided into several500 mL shake flasks with 100 mL working volume and then dosed withvarious amounts of MPA (0 μM, 20 μM, 40 μM) or acMPAG (20 μM) induplicates. After dosing, the shake flasks were cultured another 4 daysat 31° C., 5% CO₂ and 150 rpm with fed-batch process and then harvested.The supernatant was sent for PQ assay (N-glycan analysis) forafucosylation level.

The data showed that an increase in MPA dosing concentration caused anincrease in the afucosylation level, and that acMPAG dosing alsoincreased the afucosylation level to similar extent (FIG. 18).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method of altering the glycosylation pattern ofa recombinant glycoprotein produced in cell culture comprising:culturing eukaryotic cells engineered to express a recombinantglycoprotein of interest in a cell culture medium, wherein the cellculture medium is supplemented with an additive comprising mycophenolicacid; wherein the glycosylation pattern of the recombinant glycoproteinof interest is altered relative to the same recombinant glycoproteinproduced by the same cells in the same cell culture medium without theadditive; wherein the recombinant glycoprotein of interest comprises atleast a portion of: an antibody, an immunoadhesin, a Transforming GrowthFactor (TGF) beta superfamily signaling molecule, a blood clottingfactor, antibody against TNFR, antibody against growth factor receptors(HER2), TNFR:Fc, combinations thereof, or fragments thereof.
 2. Themethod of claim 1 wherein the glycosylation pattern of the recombinantglycoprotein of interest is altered to better resemble the glycosylationpattern of a reference sample of the glycoprotein.
 3. The method ofclaim 1, further comprising recovering the recombinant glycoprotein ofinterest from the cell culture.
 4. The method of claim 1, wherein thealteration of the glycosylation pattern of the recombinant glycoproteinof interest comprises an increased level of afucosylation.
 5. The methodof claim 1, wherein the alteration of the glycosylation pattern isachieved without increasing the level of α-gal.
 6. The method of claim1, wherein the alteration of the glycosylation pattern is achievedwithout reducing sialic acid levels.
 7. The method of claim 1, whereinthe cell culture comprises a growth phase and a protein productionphase, and wherein the additive is introduced into the culture mediumbefore or at the same time as the initiation of the protein productionphase.
 8. The method of claim 1, wherein the cell culture is conductedin a shake flask.
 9. The method of claim 1, wherein the cell culture isconducted in a stirred-tank bioreactor.
 10. The method of claim 9,wherein the bioreactor has a volume of between about 500 liters andabout 30,000 liters.
 11. The method of claim 1, wherein the eukaryoticcells engineered to express the recombinant glycoprotein of interest areselected from the group consisting of CHO cells, HEK cells, NSO cells,PER.C6 cells, 293 cells, HeLa cells, and MDCK cells.
 12. The method ofclaim 1, wherein the eukaryotic cells engineered to express therecombinant glycoprotein of interest are hybridoma cells.
 13. The methodof claim 1, wherein the eukaryotic cells engineered to express therecombinant glycoprotein of interest have been adapted to grow in serumfree medium, animal protein free medium or chemically defined medium.14. The method of claim 1, wherein the antibody comprises an antibody Fcregion, an antigen-binding domain of an antibody, a full antibody, achimeric antibody, a humanized antibody or human antibody, or a humanIgG1 antibody.
 15. The method of claim 1, wherein the immunoadhesincomprises a tumor necrosis factor receptor.
 16. The method of claim 1,wherein the total amount of recombinant glycoprotein produced in theadditive-supplemented cell culture medium is equal to the total amountof recombinant glycoprotein produced by the corresponding unsupplementedcell culture medium.
 17. The method of claim 1, wherein the total amountof recombinant glycoprotein produced in the additive-supplemented cellculture medium is decreased by less than 20% of the total amount ofrecombinant glycoprotein produced by the corresponding unsupplementedcell culture medium.
 18. The method of claim 1, wherein the specificproductivity of the engineered eukaryotic cells maintained in theadditive-supplemented cell culture medium is equal to the specificproductivity of the same cells maintained in the correspondingunsupplemented cell culture medium.
 19. The method of claim 1, whereinthe cell culture is a perfusion culture.
 20. The method of claim 1,wherein the supplemented cell culture medium comprises between 1 μM and50 μM mycophenolic acid.
 21. The method of claim 20, wherein themycophenolic acid is introduced into the cell culture medium as part ofa feed medium, or as one or more boli from a distinct stock solution.22. The method of claim 1, wherein the method further comprisescontrolling or modulating cell culture temperature.
 23. The method ofclaim 22, wherein the cell culture temperature is about 25 to about 42°C.
 24. The method of claim 22, wherein the mycophenolic acid isintroduced at the same time as the cell culture temperature iscontrolled or modulated.
 25. The method of claim 22, wherein themycophenolic acid is introduced at a different time than the time whenthe cell culture temperature is controlled or modulated.
 26. The methodof claim 1, wherein the method further comprises controlling ormodulating cell culture seed density.
 27. A method of altering theglycosylation pattern of a recombinant glycoprotein produced in cellculture comprising: culturing eukaryotic cells engineered to express arecombinant glycoprotein of interest in a cell culture medium, whereinthe cell culture medium is supplemented with an additive comprisingmycophenolic acid; wherein the glycosylation pattern of the recombinantglycoprotein of interest is altered relative to the same recombinantglycoprotein produced by the same cells in the same cell culture mediumwithout the additive; wherein the eukaryotic cells engineered to expressthe recombinant glycoprotein of interest are hybridoma cells.
 28. Amethod of altering the glycosylation pattern of a recombinantglycoprotein produced in cell culture comprising: culturing eukaryoticcells engineered to express a recombinant glycoprotein of interest in acell culture medium, wherein the cell culture medium is supplementedwith an additive comprising mycophenolic acid; wherein the glycosylationpattern of the recombinant glycoprotein of interest is altered relativeto the same recombinant glycoprotein produced by the same cells in thesame cell culture medium without the additive; wherein the cell cultureis a perfusion culture.